Methods and compositions for cell-proliferation-related disorders

ABSTRACT

Methods of treating and evaluating subjects having neoactive mutants are described herein.

CLAIM OF PRIORITY

This application is a continuation of U.S. Ser. No. 13/939,519, filedJul. 11, 2013, which is a continuation of U.S. Ser. No. 13/256,396,filed Nov. 29, 2011, which is a national stage application under 35U.S.C. §371 of International Application No. PCT/US2010/027253, filedMar. 12, 2010, published as International Publication No. WO 2010/105243on Sep. 16, 2010, which claims priority to U.S. Ser. No. 61/160,253,filed Mar. 13, 2009; U.S. Ser. No. 61/160,664, filed Mar. 16, 2009; U.S.Ser. No. 61/173,518, filed Apr. 28, 2009; U.S. Ser. No. 61/180,609,filed May 22, 2009; U.S. Ser. No. 61/220,543, filed Jun. 25, 2009; U.S.Ser. No. 61/227,649, filed Jul. 22, 2009; U.S. Ser. No. 61/229,689,filed Jul. 29, 2009; U.S. Ser. No. 61/253,820, filed Oct. 21, 2009; andU.S. Ser. No. 61/266,929, filed Dec. 4, 2009, the contents of each ofwhich are incorporated herein by reference.

FIELD OF THE INVENTION

The invention relates to methods and compositions for evaluating andtreating cell proliferation-related disorders, e.g., proliferativedisorders such as cancer.

BACKGROUND

Isocitrate dehydrogenase, also known as IDH, is an enzyme whichparticipates in the citric acid cycle. It catalyzes the third step ofthe cycle: the oxidative decarboxylation of isocitrate, producingalpha-ketoglutarate (α-ketoglutarate or α-KG) and CO₂ while convertingNAD+ to NADH. This is a two-step process, which involves oxidation ofisocitrate (a secondary alcohol) to oxalosuccinate (a ketone), followedby the decarboxylation of the carboxyl group beta to the ketone, formingalpha-ketoglutarate. Another isoform of the enzyme catalyzes the samereaction; however this reaction is unrelated to the citric acid cycle,is carried out in the cytosol as well as the mitochondrion andperoxisome, and uses NADP+ as a cofactor instead of NAD+.

SUMMARY OF THE INVENTION

Methods and compositions disclosed herein relate to the role played indisease by neoactive products produced by neoactive mutant enzymes,e.g., mutant metabolic pathway enzymes. The inventors have discovered,inter alia, a neoactivity associated with IDH mutants and that theproduct of the neoactivity can be significantly elevated in cancercells. Disclosed herein are methods and compositions for treating, andmethods of evaluating, subjects having or at risk for a disorder, e.g.,a cell proliferation-related disorder characterized by a neoactivity ina metabolic pathway enzyme, e.g., IDH neoactivity. Such disordersinclude e.g., proliferative disorders such as cancer. The inventors havediscovered and disclosed herein novel therapeutic agents for thetreatment of disorders, e.g., cancers, characterized by, e.g., by aneoactivity, neoactive protein, neoactive mRNA, or neoactive mutations.In embodiments a therapeutic agent reduces levels of neoactivity orneoactive product or ameliorates an effect of a neoactive product.Methods described herein also allow the identification of a subject, oridentification of a treatment for the subject, on the basis ofneaoctivity genotype or phenotype. This evaluation can allow for optimalmatching of subject with treatment, e.g., where the selection ofsubject, treatment, or both, is based on an analysis of neoactivitygenotype or phenotype. E.g., methods describe herein can allow selectionof a treatment regimen comprising administration of a novel compound,e.g., a novel compound disclosed herein, or a known compound, e.g., aknown compound not previously recommended for a selected disorder. Inembodiments the known compound reduces levels of neoactivity orneoactive product or ameliorates an effect of a neoactive product.Methods described herein can guide and provide a basis for selection andadministration of a novel compound or a known compound, or combinationof compounds, not previously recommended for subjects having a disordercharacterized by a somatic neoactive mutation in a metabolic pathwayenzyme. In embodiments the neoactive genotype or phenotype can act as abiomarker the presence of which indicates that a compound, either novel,or previously known, should be administered, to treat a disordercharacterized by a somatic neoactive mutation in a metabolic pathwayenzyme. Neoactive mutants of IDH1 having a neoactivity that results inthe production of 2-hydroxyglutarate, e.g., R-2-hydroxyglutarate andassociated disorders are discussed in detail herein. They are exemplary,but not limiting, examples of embodiments of the invention.

While not wishing to be bound by theory it is believed that the balancebetween the production and elimination of neoactive product, e.g., 2HG,e.g., R-2HG, is important in disease. Neoactive mutants, to varyingdegrees for varying mutations, increase the level of neoactive product,while other processes, e.g., in the case of 2HG, e.g., R-2HG, enzymaticdegradation of 2HG, e.g., by 2HG dehydrogenase, reduce the level ofneoactive product. An incorrect balance is associated with disease. Inembodiments, the net result of a neoactive mutation at IDH1 or IDH2result in increased levels, in affected cells, of neoactive product,2HG, e.g., R-2HG,

Accordingly, in one aspect, the invention features, a method of treatinga subject having a cell proliferation-related disorder, e.g., a disordercharacterized by unwanted cell proliferation, e.g., cancer, or aprecancerous disorder. The cell proliferation-related disorder ischaracterized by a somatic mutation in a metabolic pathway enzyme. Themutation is associated with a neoactivity that results in the productionof a neoactivity product. The method comprises: administering to thesubject a therapeutically effective amount of a therapeutic agentdescribed herein, e.g., a therapeutic agent that decreases the level ofneoactivity product encoded by a selected or mutant somatic allele,e.g., an inhibitor of a neoactivity of the metabolic pathway enzyme (theneoactive enzyme), a therapeutic agent that ameliorates an unwantedaffect of the neoactivity product, or a nucleic acid based inhibitor,e.g., a dRNA which targets the neoactive enzyme mRNA, to thereby treatthe subject.

In an embodiment the subject is a subject not having, or not diagnosedas having, 2-hydroxyglutaric aciduria.

In an embodiment the subject has a cell proliferation-related disorder,e.g., a cancer, characterized by the neoactivity of the metabolicpathway enzyme encoded by selected or mutant allele.

In an embodiment the subject has a cell proliferation-related disorder,e.g., a cancer, characterized by the product formed by the neoactivityof the metabolic pathway enzyme encoded by selected or mutant allele.

In one embodiment, the metabolic pathway is selected from a metabolicpathway leading to fatty acid biosynthesis, glycolysis, glutaminolysis,the pentose phosphate shunt, nucleotide biosynthetic pathways, or thefatty acid biosynthetic pathway.

In an embodiment the therapeutic agent is a therapeutic agent describedherein.

In an embodiment the method comprises selecting a subject on the basisof having a cancer characterized by the selected or mutant allele, theneoactivity, or an elevated level of neaoctivity product.

In an embodiment the method comprises selecting a subject on the basisof having a cancer characterized by the product formed by theneoactivity of the protein encoded by selected or mutant allele, e.g.,by the imaging and/or spectroscopic analysis, e.g., magneticresonance-based analysis, e.g., MRI (magnetic resonance imaging) and/orMRS (magnetic resonance spectroscopy), to determine the presence,distribution or level of the product of the neoactivity, e.g., in thecase of an IDH1 allele described herein, 2-hydroxyglutarate (sometimesreferred to herein as 2HG), e.g., R-2-hydroxyglutarate (sometimesreferred to herein as R-2HG).

In an embodiment the method comprises confirming or determining, e.g.,by direct examination or evaluation of the subject, or sample e.g.,tissue, product (e.g., feces, sweat, semen, exhalation, hair or nails),or bodily fluid (e.g., blood (e.g., blood plasma), urine, lymph, orcerebrospinal fluid or other sample sourced disclosed herein) therefrom,(e.g., by DNA sequencing, immuno analysis, or assay for enzymaticactivity), or receiving such information about the subject, that thecancer is characterized by the selected or mutant allele.

In an embodiment the method comprises confirming or determining, e.g.,by direct examination or evaluation of the subject, the level ofneoactivity or the level of the product of the neoactivity, or receivingsuch information about the subject. In an embodiment the presence,distribution or level of the product of the neoactivity, e.g., in thecase of an IDH1 allele described herein, 2HG, e.g., R-2HG, is determinednon-invasively, e.g., by imaging methods, e.g., by magneticresonance-based methods.

In an embodiment the method comprises administering a second anti-canceragent or therapy to the subject, e.g., surgical removal oradministration of a chemotherapeutic.

In another aspect, the invention features, a method of treating asubject having a cell proliferation-related disorder, e.g., aprecancerous disorder, or cancer. In an embodiment the subject does nothave, or has not been diagnosed as having, 2-hydroxyglutaric aciduria.The cell proliferation-related disorder is characterized by a somaticallele, e.g., a preselected allele, or mutant allele, of an IDH, e.g.,IDH1 or IDH2, which encodes a mutant IDH, e.g., IDH1 or IDH2, enzymehaving a neoactivity.

In embodiments the neoactivity is alpha hydroxy neoactivity. As usedherein, alpha hydroxy neoactivity refers to the ability to convert analpha ketone to an alpha hydroxy. In embodiments alpha hydroxyneoactivity proceeds with a reductive cofactor, e.g., NADPH or NADH. Inembodiments the alpha hydroxyl neoactivity is 2HG neoactivity. 2HGneoactivity, as used herein, refers to the ability to convert alphaketoglutarate to 2-hydroxyglutarate (sometimes referred to herein as2HG), e.g., R-2-hydroxyglutarate (sometimes referred to herein asR-2HG). In embodiments 2HG neoactivity proceeds with a reductivecofactor, e.g., NADPH or NADH. In an embodiment a neoactive enzyme,e.g., an alpha hydroxyl, e.g., a 2HG, neoactive enzyme, can act on morethan one substrate, e.g., more than one alpha hydroxy substrate.

The method comprises administering to the subject an effective amount ofa therapeutic agent of type described herein to thereby treat thesubject.

In an embodiment the therapeutic agent: results in lowering the level ofa neoactivity product, e.g., an alpha hydroxy neoactivity product, e.g.,2HG, e.g., R-2HG.

In an embodiment the method comprises administering a therapeutic agentthat lowers neoactivity, e.g., 2HG neoactivity. In an embodiment themethod comprises administering an inhibitor of a mutant IDH protein,e.g., a mutant IDH1 or mutant IDH2 protein, having a neoactivity, e.g.,alpha hydroxy neoactivity, e.g., 2HG neoactivity.

In an embodiment the therapeutic agent comprises a compound from Table24a or Table 24b or a compound having the structure of Formula (X) or(Formula (XI) described herein.

In an embodiment the therapeutic agent comprises nucleic acid-basedtherapeutic agent, e.g., a dsRNA, e.g., a dsRNA described herein.

In an embodiment the therapeutic agent is an inhibitor, e.g., apolypeptide, peptide, or small molecule (e.g., a molecule of less than1,000 daltons), or aptomer, that binds to an IDH1 mutant or wildtypesubunit and inhibits neoactivity, e.g., by inhibiting formation of adimer, e.g., a homodimer of mutant IDH1 subunits or a heterodimer of amutant and a wildtype subunit. In an embodiment the inhibitor is apolypeptide. In an embodiment the polypeptide acts as a dominantnegative with respect to the neoactivity of the mutant enzyme. Thepolypeptide can correspond to full length IDH1 or a fragment thereof.The polypeptide need not be identical with the corresponding residues ofwildtype IDH1, but in embodiments has at least 60, 70, 80, 90 or 95%homology with wildtype IDH1.

In an embodiment the therapeutic agent decreases the affinity of an IDH,e.g., IDH1 or IDH2 neoactive mutant protein for NADH, NADPH or adivalent metal ion, e.g., Mg²⁺ or Mn²⁺, or decreases the levels oravailability of NADH, NADPH or divalent metal ion, e.g., Mg²⁺ or Mn²⁺,e.g., by competing for binding to the mutant enzyme. In an embodimentthe enzyme is inhibited by replacing Mg²⁺ or Mn²⁺ with Ca²⁺.

In an embodiment the therapeutic agent is an inhibitor that reduces thelevel a neoactivity of an IDH, e.g., IDH1 or IDH2, e.g., 2HGneoactivity.

In an embodiment the therapeutic agent is an inhibitor that reduces thelevel of the product of a mutant having a neoactivity of an IDH, e.g.,IDH1 or IDH2 mutant, e.g., it reduces the level of 2HG, e.g., R-2HG.

In an embodiment the therapeutic agent is an inhibitor that:

inhibits, e.g., specifically, a neoactivity of an IDH, e.g., IDH1 orIDH2, e.g., a neoactivity described herein, e.g., 2HG neoactivity; or

inhibits both the wildtype activity and a neoactivity of an IDH, e.g.,IDH1 or IDH2, e.g., a neoactivity described herein, e.g., 2HGneoactivity.

In an embodiment the therapeutic agent is an inhibitor that is selectedon the basis that it:

inhibits, e.g., specifically, a neoactivity of an IDH, e.g., IDH1 orIDH2, e.g., a neoactivity described herein e.g., 2HG neoactivity; or

inhibits both the wildtype activity and a neoactivity of an IDH1, e.g.,IDH1 or IDH2, e.g., a neoactivity described herein, e.g., 2HGneoactivity.

In an embodiment the therapeutic agent is an inhibitor that reduces theamount of a mutant IDH, e.g., IDH1 or IDH2, protein or mRNA.

In an embodiment the therapeutic agent is an inhibitor that interactsdirectly with, e.g., it binds to, the mutant IDH, e.g., IDH1 or IDH2mRNA.

In an embodiment the therapeutic agent is an inhibitor that interactsdirectly with, e.g., it binds to, the mutant IDH, e.g., IDH1 or IDH2,protein.

In an embodiment the therapeutic agent is an inhibitor that reduces theamount of neoactive enzyme activity, e.g., by interacting with, e.g.,binding to, mutant IDH, e.g., IDH1 or IDH2, protein. In an embodimentthe inhibitor is other than an antibody.

In an embodiment the therapeutic agent is an inhibitor that is a smallmolecule and interacts with, e.g., binds, the mutant RNA, e.g., mutantIDH1 or IDH2 mRNA (e.g., mutant IDH1 mRNA).

In an embodiment the therapeutic agent is an inhibitor that interactsdirectly with, e.g., binds, either the mutant IDH, e.g., IDH1 or IDH2,protein or interacts directly with, e.g., binds, the mutant IDH mRNA,e.g., IDH1 or IDH2 mRNA.

In an embodiment the IDH is IDH1 and the neoactivity is alpha hydroxyneoactivity, e.g., 2HG neoactivity. Mutations in IDH1 associated with2HG neoactivity include mutations at residue 132, e.g., R132H, R132C,R132S, R132G, R132L, or R132V (e.g., R132H or R132C).

In an embodiment the IDH is IDH2 and the neoactivity of the IDH2 mutantis alpha hydroxy neoactivity, e.g., 2HG neoactivity. Mutations in IDH2associated with 2HG neoactivity include mutations at residue 172, e.g.,R172K, R172M, R172S, R172G, or R172W.

Treatment methods described herein can comprise evaluating a neoactivitygenotype or phenotype. Methods of obtaining and analyzing samples, andthe in vivo analysis in subjects, described elsewhere herein, e.g., inthe section entitled, “Methods of evaluating samples and/or subjects,”can be combined with this method.

In an embodiment, prior to or after treatment, the method includesevaluating the growth, size, weight, invasiveness, stage or otherphenotype of the cell proliferation-related disorder.

In an embodiment, prior to or after treatment, the method includesevaluating the IDH, e.g., IDH1 or IDH2, alpha hydroxyl neoactivitygenotype, e.g., 2HG, genotype, or alpha hydroxy neoactivity phenotype,e.g., 2HG, e.g., R-2HG, phenotype. Evaluating the alpha hydroxyl, e.g.,2HG, genotype can comprise determining if an IDH1 or IDH2 mutationhaving alpha hydroxy neoactivity, e.g., 2HG neoactivity, is present,e.g., a mutation disclosed herein having alpha hydroxy neoactivity,e.g., 2HG neoactivity. Alpha hydroxy neoactivity phenotype, e.g., 2HG,e.g., R-2HG, phenotype, as used herein, refers to the level of alphahydroxy neoactivity product, e.g., 2HG, e.g., R-2HG, level of alphahydroxy neoactivity, e.g., 2HG neoactivity, or level of mutant enzymehaving alpha hydroxy neoactivity, e.g., 2HG neoactivity (orcorresponding mRNA). The evaluation can be by a method described herein.

In an embodiment the subject can be evaluated, before or aftertreatment, to determine if the cell proliferation-related disorder ischaracterized by an alpha hydroxy neoactivity product, e.g., 2HG, e.g.,R-2HG.

In an embodiment a cancer, e.g., a glioma or brain tumor in a subject,can be analyzed, e.g., by imaging and/or spectroscopic analysis, e.g.,magnetic resonance-based analysis, e.g., MRI and/or MRS, e.g., before orafter treatment, to determine if it is characterized by presence of analpha hydroxy neoactivity product, e.g., 2HG, e.g., R-2HG.

In an embodiment the method comprises evaluating, e.g., by directexamination or evaluation of the subject, or a sample from the subject,or receiving such information about the subject, the IDH, e.g., IDH1 orIDH2, genotype, or an alpha hydroxy neoactivity product, e.g., 2HG,e.g., R-2HG phenotype of, the subject, e.g., of a cell, e.g., a cancercell, characterized by the cell proliferation-related disorder. (Asdescribed in more detail elsewhere herein the evaluation can be, e.g.,by DNA sequencing, immuno analysis, evaluation of the presence,distribution or level of an alpha hydroxy neoactivity product, e.g.,2HG, e.g., R-2HG, e.g., from spectroscopic analysis, e.g., magneticresonance-based analysis, e.g., MRI and/or MRS measurement, sampleanalysis such as serum or spinal cord fluid analysis, or by analysis ofsurgical material, e.g., by mass-spectroscopy). In embodiments thisinformation is used to determine or confirm that a proliferation-relateddisorder, e.g., a cancer, is characterized by an alpha hydroxyneoactivity product, e.g., 2HG, e.g., R-2HG. In embodiments thisinformation is used to determine or confirm that a cellproliferation-related disorder, e.g., a cancer, is characterized by anIDH, e.g., IDH1 or IDH2, allele described herein, e.g., an IDH1 allelehaving a mutation, e.g., a His, Ser, Cys, Gly, Val, Pro or Leu (e.g.,His, Ser, Cys, Gly, Val, or Leu at residue 132, more specifically, Hisor Cys, or an IDH2 allele having a mutation at residue 172, e.g., a K,M, S, G, or W.

In an embodiment, before and/or after treatment has begun, the subjectis evaluated or monitored by a method described herein, e.g., theanalysis of the presence, distribution, or level of an alpha hydroxyneoactivity product, e.g., 2HG, e.g., R-2HG, e.g., to select, diagnoseor prognose the subject, to select an inhibitor, or to evaluate responseto the treatment or progression of disease.

In an embodiment the cell proliferation-related disorder is a tumor ofthe CNS, e.g., a glioma, a leukemia, e.g., AML or ALL, e.g., B-ALL orT-ALL, prostate cancer, fibrosarcoma, paraganglioma, or myelodysplasiaor myelodysplastic syndrome (e.g., B-ALL or T-ALL, prostate cancer, ormyelodysplasia or myelodysplastic syndrome) and the evaluation is:evaluation of the presence, distribution, or level of an alpha hydroxyneoactivity product, e.g., 2HG, e.g., R-2HG; or evaluation of thepresence, distribution, or level of a neoactivity, e.g., an alphahydroxy neoactivity, e.g., 2HG neoactivity, of an IDH1 or IDH2, mutantprotein.

In an embodiment the disorder is other than a solid tumor. In anembodiment the disorder is a tumor that, at the time of diagnosis ortreatment, does not have a necrotic portion. In an embodiment thedisorder is a tumor in which at least 30, 40, 50, 60, 70, 80 or 90% ofthe tumor cells carry an IHD, e.g., IDH1 or IDH2, mutation having 2HGneoactivity, at the time of diagnosis or treatment.

In an embodiment the cell proliferation-related disorder is a cancer,e.g., a cancer described herein, characterized by an IDH1 somatic mutanthaving alpha hydroxy neoactivity, e.g., 2HG neoactivity, e.g., a mutantdescribed herein. In an embodiment the tumor is characterized byincreased levels of an alpha hydroxy neoactivity product, 2HG, e.g.,R-2HG, as compared to non-diseased cells of the same type.

In an embodiment the method comprises selecting a subject having aglioma, on the basis of the cancer being characterized by unwanted(i.e., increased) levels of an alpha hydroxy neoactivity, product, e.g.,2HG, e.g., R-2HG.

In an embodiment the cell proliferation-related disorder is a tumor ofthe CNS, e.g., a glioma, e.g., wherein the tumor is characterized by anIDH1 somatic mutant having alpha hydroxy neoactivity, e.g., 2HGneoactivity, e.g., a mutant described herein. Gliomas include astrocytictumors, oligodendroglial tumors, oligoastrocytic tumors, anaplasticastrocytomas, and glioblastomas. In an embodiment the tumor ischaracterized by increased levels of an alpha hydroxy neoactivityproduct, e.g., 2HG, e.g., R-2HG, as compared to non-diseased cells ofthe same type. E.g., in an embodiment, the IDH1 allele encodes an IDH1having other than an Arg at residue 132. E.g., the allele encodes His,Ser, Cys, Gly, Val, Pro or Leu (e.g., His, Ser, Cys, Gly, Val, or Leu),or any residue described in Yan et al., at residue 132, according to thesequence of SEQ ID NO:8 (see also FIG. 21). In an embodiment the alleleencodes an IDH1 having His at residue 132. In an embodiment the alleleencodes an IDH1 having Ser at residue 132.

In an embodiment the IDH1 allele has an A (or any other nucleotide otherthan C) at nucleotide position 394, or an A (or any other nucleotideother than G) at nucleotide position 395. In an embodiment the allele isa C394A, a C394G, a C394T, a G395C, a G395T or a G395A mutation;specifically a C394A or a G395A mutation according to the sequence ofSEQ ID NO:5.

In an embodiment the method comprises selecting a subject having aglioma, wherein the cancer is characterized by having an IDH1 alleledescribed herein, e.g., an IDH1 allele having His, Ser, Cys, Gly, Val,Pro or Leu at residue 132 (SEQ ID NO:8), more specifically His, Ser,Cys, Gly, Val, or Leu; or His or Cys.

In an embodiment the method comprises selecting a subject having aglioma, on the basis of the cancer being characterized by an IDH1 alleledescribed herein, e.g., an IDH1 allele having His, Ser, Cys, Gly, Val,Pro or Leu at residue 132 (SEQ ID NO:8), more specifically His, Ser,Cys, Gly, Val, or Leu; or His or Cys.

In an embodiment the method comprises selecting a subject having aglioma, on the basis of the cancer being characterized by increasedlevels of an alpha hydroxy neoactivity, product, e.g., 2HG, e.g., R-2HG.

In an embodiment the method comprises selecting a subject having afibrosarcoma or paraganglioma wherein the cancer is characterized byhaving an IDH1 allele described herein, e.g., an IDH1 allele having Cysat residue 132 (SEQ ID NO:8).

In an embodiment the method comprises selecting a subject having afibrosarcoma or paraganglioma, on the basis of the cancer beingcharacterized by an IDH1 allele described herein, e.g., an IDH1 allelehaving Cys at residue 132 (SEQ ID NO:8).

In an embodiment the method comprises selecting a subject having afibrosarcoma or paraganglioma, on the basis of the cancer beingcharacterized by increased levels of an alpha hydroxy neoactivity,product, e.g., 2HG, e.g., R-2HG.

In an embodiment the cell proliferation-related disorder is localized ormetastatic prostate cancer, e.g., prostate adenocarcinoma, e.g., whereinthe cancer is characterized by an IDH1 somatic mutant having alphahydroxy neoactivity, e.g., 2HG neoactivity, e.g., a mutant describedherein. In an embodiment the cancer is characterized by increased levelsof an alpha hydroxy neoactivity product, e.g., 2HG, e.g., R-2HG, ascompared to non-diseased cells of the same type.

E.g., in an embodiment, the IDH1 allele encodes an IDH1 having otherthan an Arg at residue 132. E.g., the allele encodes His, Ser, Cys, Gly,Val, Pro or Leu, or any residue described in Kang et al, 2009, Int. J.Cancer, 125: 353-355 at residue 132, according to the sequence of SEQ IDNO:8 (see also FIG. 21) (e.g., His, Ser, Cys, Gly, Val, or Leu). In anembodiment the allele encodes an IDH1 having His or Cys at residue 132.

In an embodiment the IDH1 allele has a T (or any other nucleotide otherthan C) at nucleotide position 394, or an A (or any other nucleotideother than G) at nucleotide position 395. In an embodiment the allele isa C394T or a G395A mutation according to the sequence of SEQ ID NO:5.

In an embodiment the method comprises selecting a subject havingprostate cancer, e.g., prostate adenocarcinoma, wherein the cancer ischaracterized by an IDH1 allele described herein, e.g., an IDH1 allelehaving His or Cys at residue 132 (SEQ ID NO:8).

In an embodiment the method comprises selecting a subject havingprostate cancer, e.g., prostate adenocarcinoma, on the basis of thecancer being characterized by an IDH1 allele described herein, e.g., anIDH1 allele having His or Cys at residue 132 (SEQ ID NO:8).

In an embodiment the method comprises selecting a subject havingprostate cancer, on the basis of the cancer being characterized byincreased levels of an alpha hydroxy neoactivity product, e.g., 2HG,e.g., R-2HG.

In an embodiment the cell proliferation-related disorder is ahematological cancer, e.g., a leukemia, e.g., AML, or ALL, wherein thehematological cancer is characterized by an IDH1 somatic mutant havingalpha hydroxy neoactivity, e.g., 2HG neoactivity, e.g., a mutantdescribed herein. In an embodiment the cancer is characterized byincreased levels of an alpha hydroxy neoactivity product, e.g., 2HG,e.g., R-2HG, as compared to non-diseased cells of the same type.

In an embodiment the cell proliferation-related disorder is acutelymphoblastic leukemia (e.g., an adult or pediatric form), e.g., whereinthe acute lymphoblastic leukemia (sometimes referred to herein as ALL)is characterized by an IDH1 somatic mutant having alpha hydroxyneoactivity, e.g., 2HG neoactivity, e.g., a mutant described herein. TheALL can be, e.g., B-ALL or T-ALL. In an embodiment the cancer ischaracterized by increased levels of 2 an alpha hydroxy neoactivityproduct, e.g., HG, e.g., R-2HG, as compared to non-diseased cells of thesame type. E.g., in an embodiment, the IDH1 allele is an IDH1 havingother than an Arg at residue 132 (SEQ ID NO:8). E.g., the allele encodesHis, Ser, Cys, Gly, Val, Pro or Leu, or any residue described in Kang etal., at residue 132, according to the sequence of SEQ ID NO:8 (see alsoFIG. 21), more specifically His, Ser, Cys, Gly, Val, or Leu. In anembodiment the allele encodes an IDH1 having Cys at residue 132.

In an embodiment the IDH1 allele has a T (or any other nucleotide otherthan C) at nucleotide position 394. In an embodiment the allele is aC394T mutation according to the sequence of SEQ ID NO:5.

In an embodiment the method comprises selecting a subject having ALL,e.g., B-ALL or T-ALL, characterized by an IDH1 allele described herein,e.g., an IDH1 allele having Cys at residue 132 according to the sequenceof SEQ ID NO:8.

In an embodiment the method comprises selecting a subject ALL, e.g.,B-ALL or T-ALL, on the basis of cancer being characterized by having anIDH1 allele described herein, e.g., an IDH1 allele having Cys at residue132 (SEQ ID NO:8).

In an embodiment the method comprises selecting a subject having ALL,e.g., B-ALL or T-ALL, on the basis of the cancer being characterized byincreased levels of an alpha hydroxy neoactivity product, e.g., 2HG,e.g., R-2HG.

In an embodiment the cell proliferation-related disorder is acutemyelogenous leukemia (e.g., an adult or pediatric form), e.g., whereinthe acute myelogenous leukemia (sometimes referred to herein as AML) ischaracterized by an IDH1 somatic mutant having alpha hydroxyneoactivity, e.g., 2HG neoactivity, e.g., a mutant described herein. Inan embodiment the cancer is characterized by increased levels of analpha hydroxy neoactivity product, e.g., 2HG, e.g., R-2HG, as comparedto non-diseased cells of the same type. E.g., in an embodiment, the IDH1allele is an IDH1 having other than an Arg at residue 132 (SEQ ID NO:8).E.g., the allele encodes His, Ser, Cys, Gly, Val, Pro or Leu, or anyresidue described in Kang et al., at residue 132, according to thesequence of SEQ ID NO:8 (see also FIG. 21). In an embodiment the alleleencodes an IDH1 having Cys, His or Gly at residue 132, morespecifically, Cys at residue 132.

In an embodiment the IDH1 allele has a T (or any other nucleotide otherthan C) at nucleotide position 394. In an embodiment the allele is aC394T mutation according to the sequence of SEQ ID NO:5.

In an embodiment the method comprises selecting a subject having acutemyelogenous lymphoplastic leukemia (AML) characterized by an IDH1 alleledescribed herein, e.g., an IDH1 allele having Cys, His, or Gly atresidue 132 according to the sequence of SEQ ID NO:8, more specifically,Cys at residue 132.

In an embodiment the method comprises selecting a subject having acutemyelogenous lymphoplastic leukemia (AML) on the basis of cancer beingcharacterized by having an IDH1 allele described herein, e.g., an IDH1allele having Cys, His, or Gly at residue 132 (SEQ ID NO:8), morespecifically, Cys at residue 132.

In an embodiment the method comprises selecting a subject having acutemyelogenous lymphoplastic leukemia (AML), on the basis of the cancerbeing characterized by increased levels of an alpha hydroxy neoactivityproduct, e.g., 2HG, e.g., R-2HG.

In an embodiment the method further comprises evaluating the subject forthe presence of a mutation in the NRAS or NPMc gene.

In an embodiment the cell proliferation-related disorder ismyelodysplasia or myelodysplastic syndrome, e.g., wherein themyelodysplasia or myelodysplastic syndrome is characterized by having anIDH1 somatic mutant having alpha hydroxy neoactivity, e.g., 2HGneoactivity, e.g., a mutant described herein. In an embodiment thedisorder is characterized by increased levels of an alpha hydroxyneoactivity product, e.g., 2HG, e.g., R-2HG, as compared to non-diseasedcells of the same type. E.g., in an embodiment, the IDH1 allele is anIDH1 having other than an Arg at residue 132 (SEQ ID NO:8). E.g., theallele encodes His, Ser, Cys, Gly, Val, Pro or Leu, or any residuedescribed in Kang et al., according to the sequence of SEQ ID NO:8 (seealso FIG. 21), more specifically His, Ser, Cys, Gly, Val, or Leu. In anembodiment the allele encodes an IDH1 having Cys at residue 132.

In an embodiment the IDH1 allele has a T (or any other nucleotide otherthan C) at nucleotide position 394. In an embodiment the allele is aC394T mutation according to the sequence of SEQ ID NO:5.

In an embodiment the method comprises selecting a subject havingmyelodysplasia or myelodysplastic syndrome characterized by an IDH1allele described herein, e.g., an IDH1 allele having Cys, His, or Gly atresidue 132 according to the sequence of SEQ ID NO:8, more specifically,Cys at residue 132.

In an embodiment the method comprises selecting a subject havingmyelodysplasia or myelodysplastic syndrome on the basis of cancer beingcharacterized by having an IDH1 allele described herein, e.g., an IDH1allele having Cys, His, or Gly at residue 132 (SEQ ID NO:8), morespecifically, Cys at residue 132.

In an embodiment the method comprises selecting a subject havingmyelodysplasia or myelodysplastic syndrome, on the basis of the cancerbeing characterized by increased levels of an alpha hydroxy neoactivityproduct, e.g., 2HG, e.g., R-2HG.

In an embodiment the cell proliferation-related disorder is a glioma,characterized by a mutation, or preselected allele, of IDH2 associatedwith an alpha hydroxy neoactivity, e.g., 2HG neoactivity. E.g., in anembodiment, the IDH2 allele encodes an IDH2 having other than an Arg atresidue 172. E.g., the allele encodes Lys, Gly, Met, Trp, Thr, Ser, orany residue described in described in Yan et al., at residue 172,according to the sequence of SEQ ID NO:10 (see also FIG. 22), morespecifically Lys, Gly, Met, Trp, or Ser. In an embodiment the alleleencodes an IDH2 having Lys at residue 172. In an embodiment the alleleencodes an IDH2 having Met at residue 172.

In an embodiment the method comprises selecting a subject having aglioma, wherein the cancer is characterized by having an IDH2 alleledescribed herein, e.g., an IDH2 allele having Lys, Gly, Met, Trp, Thr,or Ser at residue 172 (SEQ ID NO:10), more specifically Lys, Gly, Met,Trp, or Ser; or Lys or Met.

In an embodiment the method comprises selecting a subject having aglioma, on the basis of the cancer being characterized by an IDH2 alleledescribed herein, e.g., an IDH2 allele having Lys, Gly, Met, Trp, Thr,or Ser at residue 172 (SEQ ID NO:10), more specifically Lys, Gly, Met,Trp, or Ser; or Lys or Met.

In an embodiment the method comprises selecting a subject having aglioma, on the basis of the cancer being characterized by increasedlevels of an alpha hydroxy neoactivity product, e.g., 2HG, e.g., R-2HG.

In an embodiment the cell proliferation-related disorder is a prostatecancer, e.g., prostate adenocarcinoma, characterized by a mutation, orpreselected allele, of IDH2 associated with an alpha hydroxyneoactivity, e.g., 2HG neoactivity. E.g., in an embodiment, the IDH2allele encodes an IDH2 having other than an Arg at residue 172. E.g.,the allele encodes Lys, Gly, Met, Trp, Thr, Ser, or any residuedescribed in described in Yan et al., at residue 172, according to thesequence of SEQ ID NO:10 (see also FIG. 22), more specifically Lys, Gly,Met, Trp, or Ser. In an embodiment the allele encodes an IDH2 having Lysat residue 172. In an embodiment the allele encodes an IDH2 having Metat residue 172.

In an embodiment the method comprises selecting a subject having aprostate cancer, e.g., prostate adenocarcinoma, wherein the cancer ischaracterized by having an IDH2 allele described herein, e.g., an IDH2allele having Lys or Met at residue 172 (SEQ ID NO:10).

In an embodiment the method comprises selecting a subject having aprostate cancer, e.g., prostate adenocarcinoma, on the basis of thecancer being characterized by an IDH2 allele described herein, e.g., anIDH2 allele having Lys or Met at residue 172 (SEQ ID NO:10).

In an embodiment the method comprises selecting a subject having aprostate cancer, e.g., prostate adenocarcinoma, on the basis of thecancer being characterized by increased levels of an alpha hydroxyneoactivity product, e.g., 2HG, e.g., R-2HG.

In an embodiment the cell proliferation-related disorder is ALL, e.g.,B-ALL or T-ALL, characterized by a mutation, or preselected allele, ofIDH2 associated with an alpha hydroxy neoactivity, e.g., 2HGneoactivity. E.g., in an embodiment, the IDH2 allele encodes an IDH2having other than an Arg at residue 172. E.g., the allele encodes Lys,Gly, Met, Trp, Thr, Ser, or any residue described in described in Yan etal., at residue 172, according to the sequence of SEQ ID NO:10 (see alsoFIG. 22). In an embodiment the allele encodes an IDH2 having Lys atresidue 172. In an embodiment the allele encodes an IDH2 having Met atresidue 172.

In an embodiment the method comprises selecting a subject having ALL,e.g., B-ALL or T-ALL, wherein the cancer is characterized by having anIDH2 allele described herein, e.g., an IDH2 allele having Lys or Met atresidue 172 (SEQ ID NO:10).

In an embodiment the method comprises selecting a subject having ALL,e.g., B-ALL or T-ALL, on the basis of the cancer being characterized byan IDH2 allele described herein, e.g., an IDH2 allele having Lys or Metat residue 172 (SEQ ID NO:10).

In an embodiment the method comprises selecting a subject having ALL,e.g., B-ALL or T-ALL, on the basis of the cancer being characterized byincreased levels of an alpha hydroxy neoactivity product, e.g., 2HG,e.g., R-2HG.

In an embodiment the cell proliferation-related disorder is AML,characterized by a mutation, or preselected allele, of IDH2 associatedwith an alpha hydroxy neoactivity, e.g., 2HG neoactivity. E.g., in anembodiment, the IDH2 allele encodes an IDH2 having other than an Arg atresidue 172. E.g., the allele encodes Lys, Gly, Met, Trp, Thr, Ser, orany residue described in described in Yan et al., at residue 172,according to the sequence of SEQ ID NO:10 (see also FIG. 22), morespecifically Lys, Gly, Met, or Ser. In an embodiment the allele encodesan IDH2 having Lys at residue 172. In an embodiment the allele encodesan IDH2 having Met at residue 172. In an embodiment the allele encodesan IDH2 having Gly at residue 172.

In an embodiment the method comprises selecting a subject having AML,wherein the cancer is characterized by having an IDH2 allele describedherein, e.g., an IDH2 allele having Lys, Gly or Met at residue 172 (SEQID NO:10), more specifically Lys or Met.

In an embodiment the method comprises selecting a subject having AML, onthe basis of the cancer being characterized by an IDH2 allele describedherein, e.g., an IDH2 allele having Lys, Gly, or Met at residue 172 (SEQID NO:10), more specifically Lys or Met.

In an embodiment the method comprises selecting a subject having AML, onthe basis of the cancer being characterized by increased levels of analpha hydroxy neoactivity product, e.g., 2HG, e.g., R-2HG.

In an embodiment the cell proliferation-related disorder ismyelodysplasia or myelodysplastic syndrome, characterized by a mutation,or preselected allele, of IDH2. E.g., in an embodiment, the IDH2 alleleencodes an IDH2 having other than an Arg at residue 172. E.g., theallele encodes Lys, Gly, Met, Trp, Thr, Ser, or any residue described indescribed in Yan et al., at residue 172, according to the sequence ofSEQ ID NO:10 (see also FIG. 22), more specifically Lys, Gly, Met, Trp orSer. In an embodiment the allele encodes an IDH2 having Lys at residue172. In an embodiment the allele encodes an IDH2 having Met at residue172. In an embodiment the allele encodes an IDH2 having Gly at residue172.

In an embodiment the method comprises selecting a subject havingmyelodysplasia or myelodysplastic syndrome, wherein the cancer ischaracterized by having an IDH2 allele described herein, e.g., an IDH2allele having Lys, Gly, or Met at residue 172 (SEQ ID NO:10), inspecific embodiments, Lys or Met.

In an embodiment the method comprises selecting a subject havingmyelodysplasia or myelodysplastic syndrome, on the basis of the cancerbeing characterized by an IDH2 allele described herein, e.g., an IDH2allele having Lys, Gly, or Met at residue 172 (SEQ ID NO:10), inspecific embodiments, Lys or Met.

In an embodiment the method comprises selecting a subject havingmyelodysplasia or myelodysplastic syndrome, on the basis of the cancerbeing characterized by increased levels of an alpha hydroxy neoactivityproduct, e.g., 2HG, e.g., R-2HG.

In an embodiment a product of the neoactivity is 2HG (e.g., R-2HG) whichacts as a metabolite. In another embodiment a product of the neoactivityis 2HG (e.g., R-2HG) which acts as a toxin, e.g., a carcinogen.

In some embodiments, the methods described herein can result in reducedside effects relative to other known methods of treating cancer.

Therapeutic agents and methods of subject evaluation described hereincan be combined with other therapeutic mocalities, e.g., with art-knowntreatments.

In an embodiment the method comprises providing a second treatment, tothe subject, e.g., surgical removal, irradiation or administration of achemotherapeutitc agent, e.g., an administration of an alkylating agent.Administration (or the establishment of therapeutic levels) of thesecond treatment can: begin prior to the beginning or treatment with (orprior to the establishment of therapeutic levels of) the inhibitor;begin after the beginning or treatment with (or after the establishmentof therapeutic levels of) the inhibitor, or can be administeredconcurrently with the inhibitor, e.g., to achieve therapeutic levels ofboth concurrently.

In an embodiment the cell proliferation-related disorder is a CNS tumor,e.g., a glioma, and the second therapy comprises administration of oneor more of: radiation; an alkylating agent, e.g., temozolomide, e.g.,Temoader®, or BCNU; or an inhibitor of HER1/EGFR tyrosine kinase, e.g.,erlotinib, e.g., Tarceva®.

The second therapy, e.g., in the case of glioma, can compriseimplantation of BCNU or carmustine in the brain, e.g., implantation of aGliadel® wafer.

The second therapy, e.g., in the case of glioma, can compriseadministration of imatinib, e.g., Gleevec®.

In an embodiment the cell proliferation-related disorder is prostatecancer and the second therapy comprises one or more of: androgenablation; administration of a microtubule stabilizer, e.g., docetaxol,e.g., Taxotere®; or administration of a topoisomerase II inhibitor,e.g., mitoxantrone.

In an embodiment the cell proliferation-related disorder is ALL, e.g.,B-ALL or T-ALL, and the second therapy comprises one or more of:

induction phase treatment comprising the administration of one or moreof: a steroid; an inhibitor of microtubule assembly, e.g., vincristine;an agent that reduces the availability of asparagine, e.g.,asparaginase; an anthracycline; or an antimetabolite, e.g.,methotrexate, e.g., intrathecal methotrexate, or 6-mercaptopurine;

consolidation phase treatment comprising the administration of one ormore of: a drug listed above for the induction phase; an antimetabolite,e.g., a guanine analog, e.g., 6-thioguanine; an alkylating agent, e.g.,cyclophosphamide; an anti-metabolite, e.g., AraC or cytarabine; or aninhibitor of topoisomerase I, e.g., etoposide; or

maintenance phase treatment comprising the administration of one or moreof the drugs listed above for induction or consolidation phasetreatment.

In an embodiment the cell proliferation-related disorder is AML and thesecond therapy comprises administration of one or more of: an inhibitorof topoisomerase II, e.g., daunorubicin, idarubicin, topotecan ormitoxantrone; an inhibitor of topoisomerase I, e.g., etoposide; or ananti-metabolite, e.g., AraC or cytarabine.

In another aspect, the invention features, a method of evaluating, e.g.diagnosing, a subject, e.g., a subject not having, or not diagnosed ashaving, 2-hydroxyglutaric aciduria. The method comprises analyzing aparameter related to the neoactivity genotype or phenotype of thesubject, e.g., analyzing one or more of:

a) the presence, distribution, or level of a neoactive product, e.g.,the product of an alpha hydroxy neoactivity, e.g., 2HG, e.g., R-2HG,e.g., an increased level of product, 2HG, e.g., R-2HG (as used herein,an increased level of a product of an alpha hydroxy neoactivity, e.g.,2HG, e.g., R-2HG, or similar term, e.g., an increased level of neoactiveproduct or neoactivity product, means increased as compared with areference, e.g., the level seen in an otherwise similar cell lacking theIDH mutation, e.g., IDH1 or IDH2 mutation, or in a tissue or productfrom a subject noth having the mutation (the terms increased andelevated as refered to the level of a product of alpha hydroxylneoactivity as used herein, are used interchangably);

b) the presence, distribution, or level of a neoactivity, e.g., alphahydroxy neoactivity, e.g., 2HG neoactivity, of an IDH1 or IDH2, mutantprotein;

c) the presence, distribution, or level of a neoactive mutant protein,e.g., an IDH, e.g., an IDH1 or IDH2, mutant protein which has aneoactivity, e.g., alpha hydroxy neoactivity, e.g., 2HG neoactivity, ora corresponding RNA; or

d) the presence of a selected somatic allele or mutation conferringneoactivity, e.g., an IDH, e.g., IDH1 or IDH2, which encodes a proteinwith a neoactivity, e.g., alpha hydroxy neoactivity, e.g., 2HGneoactivity, e.g., an allele disclosed herein, in cells characterized bya cell proliferation-related disorder from the subject, therebyevaluating the subject.

In an embodiment analyzing comprises performing a procedure, e.g., atest, to provide data or information on one or more of a-d, e.g.,performing a method which results in a physical change in a sample, inthe subject, or in a device or reagent used in the analysis, or whichresults in the formation of an image representative of the data. Methodsof obtaining and analyzing samples, and the in vivo analysis insubjects, described elsewhere herein, e.g., in the section entitled,“Methods of evaluating samples and/or subjects,” can be combined withthis method. In another embodiment analyzing comprises receiving data orinformation from such test from another party. In an embodiment theanalyzing comprises receiving data or information from such test fromanother party and, the method comprises, responsive to that data orinformation, administering a treatment to the subject.

As described herein, the evaluation can be used in a number ofapplications, e.g., for diagnosis, prognosis, staging, determination oftreatment efficacy, patent selection, or drug selection.

Thus, in an embodiment method further comprises, e.g., responsive to theanalysis of one or more of a-d:

diagnosing the subject, e.g., diagnosing the subject as having a cellproliferation-related disorder, e.g., a disorder characterized byunwanted cell proliferation, e.g., cancer, or a precancerous disorder;

staging the subject, e.g., determining the stage of a cellproliferation-related disorder, e.g., a disorder characterized byunwanted cell proliferation, e.g., cancer, or a precancerous disorder;

providing a prognosis for the subject, e.g., providing a prognosis for acell proliferation-related disorder, e.g., a disorder characterized byunwanted cell proliferation, e.g., cancer, or a precancerous disorder;

determining the efficacy of a treatment, e.g., the efficacy of achemotherapeutic agent, irradiation or surgery;

determining the efficacy of a treatment with a therapeutic agent, e.g.,an inhibitor, described herein;

selecting the subject for a treatment for a cell proliferation-relateddisorder, e.g., a disorder characterized by unwanted cell proliferation,e.g., cancer, or a precancerous disorder. The selection can be based onthe need for a reduction in neoactivity or on the need for ameliorationof a condition associated with or resulting from neoactivity. Forexample, if it is determined that the subject has a cellproliferation-related disorder, e.g., e.g., cancer, or a precancerousdisorder characterized by increased levels of an alpha hydroxyneoactivity product, e.g., 2HG, e.g., R-2HG, or by a mutant IDH1 orIDH2, having alpha hydroxyl neoactivity, e.g., 2HG, neaoctivity,selecting the subject for treatment with a therapeutic agent describedherein, e.g., an inhibitor (e.g., a small molecule or a nucleicacid-based inhibitor) of the neoactivity of that mutant (e.g.,conversion of alpha-ketoglutarate to 2HG, e.g., R-2HG);

correlating the analysis with an outcome or a prognosis;

providing a value for an analysis on which the evaluation is based,e.g., the value for a parameter correlated to the presence,distribution, or level of an alpha hydroxyl neoactivity product, e.g.,2HG, e.g., R-2HG;

providing a recommendation for treatment of the subject; or

memorializing a result of, or output from, the method, e.g., ameasurement made in the course of performing the method, and optionallytransmitting the memorialization to a party, e.g., the subject, ahealthcare provider, or an entity that pays for the subject's treatment,e.g., a government, insurance company, or other third party payer.

As described herein, the evaluation can provide information on which anumber of decisions or treatments can be based.

Thus, in an embodiment the result of the evaluation, e.g., an increasedlevel of an alpha hydroxyl neoactivity product, e.g., 2HG, e.g., R-2HG,the presence of an IDH, e.g., IDH1 or IDH2, neoactivity, e.g., alphahydroxyl neoactivity, e.g., 2HG neoactivity, the presence of an IDH,e.g., IDH1 or IDH2, mutant protein (or corresponding RNA) which hasalpha hydroxyl neoactivity, e.g., 2HG neoactivity, the presence of amutant allele of IDH, e.g., IDH1 or IDH2, having alpha hydroxylneoactivity, 2HG neoactivity, e.g., an allele disclosed herein, isindicative of:

a cell proliferation-related disorder, e.g., cancer, e.g., it isindicative of a primary or metastatic lesion;

the stage of a cell proliferation-related disorder;

a prognosis or outcome for a cell proliferation-related disorder, e.g.,it is indicative of a less aggressive form of the disorder, e.g.,cancer. E.g., in the case of glioma, presence of an alpha hydroxylneoactivity product, e.g., 2HG, e.g., R-2HG, can indicate a lessaggressive form of the cancer;

the efficacy of a treatment, e.g., the efficacy of a chemotherapeuticagent, irradiation or surgery;

the need of a therapy disclosed herein, e.g., inhibition a neoactivityof an IDH, e.g., IDH1 or IDH2, neoactive mutant described herein. In anembodiment relatively higher levels (or the presence of the mutant) iscorrelated with need of inhibition a neoactivity of an IDH, e.g., IDH1or IDH2, mutant described herein; or

responsiveness to a treatment. The result can be used as a noninvasivebiomarker for clinical response. E.g., elevated levels can be predictiveon better outcome in glioma patients (e.g., longer life expectancy).

As described herein, the evaluation can provide for the selection of asubject.

Thus, in an embodiment the method comprises, e.g., responsive to theanalysis of one or more of a-d, selecting a subject, e.g., for atreatment. The subject can be selected on a basis described herein,e.g., on the basis of:

said subject being at risk for, or having, higher than normal levels ofan alpha hydroxy neoactivity product, e.g., 2-hydroxyglurarate (e.g.,R-2HG) in cell having a cell proliferation-related disorder, e.g., aleukemia such as AML or ALL, e.g., B-ALL or T-ALL, or a tumor lesion,e.g., a glioma or a prostate tumor;

said subject having a proliferation-related disorder characterized by aselected IDH, e.g., IDH1 or IDH2 allele, e.g., an IDH1 or IDH2 mutation,having alpha hydroxyl neoactivity, e.g., 2HG neoactivity;

said subject having a selected IDH allele, e.g., a selected IDH1 or IDH2allele;

having alpha hydroxyl neoactivity, e.g., 2HG neoactivity;

said subject having a proliferation-related disorder;

said subject being in need of, or being able to benefit from, atherapeutic agent of a type described herein;

said subject being in need of, or being able to benefit from, a compoundthat inhibits alpha hydroxyl neoactivity, e.g., 2HG neoactivity;

said subject being in need of, or being able to benefit from, a compoundthat lowers the level of an alpha hydroxyl neoactivity product, e.g.,2HG, e.g., R-2HG.

In an embodiment evaluation comprises selecting the subject, e.g., fortreatment with an anti-neoplastic agent, on the establishment of, ordetermination that, the subject has increased alpha hydroxyl neoactivityproduct, e.g., 2HG, e.g., R-2HG, or increased alpha hydroxylneoactivity, e.g., 2HG neoactivity, or that the subject is in need ofinhibition of a neoactivity of an IDH, e.g., IDH1 or IDH2, mutantdescribed herein.

As described herein, the evaluations provided for by methods describedherein allow the selection of optimal treatment regimens.

Thus, in an embodiment the method comprises, e.g., responsive to theanalysis of one or more of a-d, selecting a treatment for the subject,e.g., selecting a treatment on a basis disclosed herein. The treatmentcan be the administration of a therapeutic agent disclosed herein. Thetreatment can be selected on the basis that:

it us useful in treating a disorder characterized by one or more ofalpha hydroxyl neoactivity, e.g., 2HG neoactivity, an IDH1 or IDH2,mutant protein having alpha hydroxyl neoactivity, e.g., 2HG neoactivity(or a corresponding RNA);

it is useful in treating a disorder characterized by a selected somaticallele or mutation of an IDH, e.g., IDH1 or IDH2, which encodes aprotein with alpha hydroxyl neoactivity, e.g., 2HG neoactivity, e.g., anallele disclosed herein, in cells characterized by a cellproliferation-related disorder from the subject;

it reduces the level of an alpha hydroxyl neoactivity product, e.g.,2HG, e.g., R-2HG;

it reduces the level of alpha hydroxyl neoactivity, e.g., 2HGneoactivity.

In an embodiment evaluation comprises selecting the subject, e.g., fortreatment.

In embodiments the treatment is the administration of a therapeuticagent described herein.

The methods can also include treating a subject, e.g, with a treatmentselected in response to, or on the basis of, an evaluation made in themethod.

Thus, in an embodiment the method comprises, e.g., responsive to theanalysis of one or more of a-d, administering a treatment to thesubject, e.g., the administration of a therapeutic agent of a typedescribed herein.

In an embodiment the therapeutic agent comprises a compound from Table24a or Table 24b or a compound having the structure of Formula (X) or(XI) described below.

In an embodiment the therapeutic agent comprises nucleic acid, e.g.,dsRNA, e.g., a dsRNA described herein.

In an embodiment the therapeutic agent is an inhibitor, e.g., apolypeptide, peptide, or small molecule (e.g., a molecule of less than1,000 daltons), or aptomer, that binds to an IDH1 or IDH2 mutant (e.g.,an aptomer that binds to an IDH1 mutant) or wildtype subunit andinhibits neoactivity, e.g., by inhibiting formation of a dimer, e.g., ahomodimer of mutant IDH1 or IDH2 subunits (e.g., a homodimer of mutantIDH1 subunits) or a heterodimer of a mutant and a wildtype subunit. Inan embodiment the inhibitor is a polypeptide. In an embodiment thepolypeptide acts as a dominant negative with respect to the neoactivityof the mutant enzyme. The polypeptide can correspond to full length IDH1or IDH2 or a fragment thereof (e.g., the polypeptide corresponds to fulllength IDH1 or a fragment thereof). The polypeptide need not beidentical with the corresponding residues of wildtype IDH1 or IDH2(e.g., wildtype IDH1), but in embodiments has at least 60, 70, 80, 90 or95% homology with wildtype IDH1 or IDH2 (e.g., wildtype IDH1).

In an embodiment the therapeutic agent decreases the affinity of an IDH,e.g., IDH1 or IDH2 neoactive mutant protein for NADH, NADPH or adivalent metal ion, e.g., Mg²⁺ or Mn²⁺, or decreases the levels oravailability of NADH, NADPH or divalent metal ion, e.g., Mg²⁺ or Mn²⁺,e.g., by competing for binding to the mutant enzyme. In an embodimentthe enzyme is inhibited by replacing Mg²⁺ or Mn²⁺ with Ca²⁺.

In an embodiment the therapeutic agent is an inhibitor that reduces thelevel a neoactivity of an IDH, e.g., IDH1 or IDH2, e.g., 2HGneoactivity.

In an embodiment the therapeutic agent is an inhibitor that reduces thelevel of the product of a mutant having a neoactivity of an IDH, e.g.,IDH1 or IDH2 mutant, e.g., it reduces the level of 2HG, e.g., R-2HG.

In an embodiment the therapeutic agent is an inhibitor that:

inhibits, e.g., specifically, a neoactivity of an IDH, e.g., IDH1 orIDH2, e.g., a neoactivity described herein, e.g., 2HG neoactivity; or

inhibits both the wildtype activity and a neoactivity of an IDH, e.g.,IDH1 or IDH2, e.g., a neoactivity described herein, e.g., 2HGneoactivity.

In an embodiment the therapeutic agent is an inhibitor that is selectedon the basis that it:

inhibits, e.g., specifically, a neoactivity of an IDH, e.g., IDH1 orIDH2, e.g., a neoactivity described herein e.g., 2HG neoactivity; or

inhibits both the wildtype activity and a neoactivity of an IDH1, e.g.,IDH1 or IDH2, e.g., a neoactivity described herein, e.g., 2HGneoactivity.

In an embodiment the therapeutic agent is an inhibitor that reduces theamount of a mutant IDH, e.g., IDH1 or IDH2, protein or mRNA.

In an embodiment the therapeutic agent is an inhibitor that interactsdirectly with, e.g., it binds to, the mutant IDH, e.g., IDH1 or IDH2mRNA.

In an embodiment the therapeutic agent is an inhibitor that interactsdirectly with, e.g., it binds to, the mutant IDH, e.g., IDH1 or IDH2,protein.

In an embodiment the therapeutic agent is an inhibitor that reduces theamount of neoactive enzyme activity, e.g., by interacting with, e.g.,binding to, mutant IDH, e.g., IDH1 or IDH2, protein. In an embodimentthe inhibitor is other than an antibody.

In an embodiment the therapeutic agent is an inhibitor that is a smallmolecule and interacts with, e.g., binds, the mutant RNA, e.g., mutantIDH1 mRNA.

In an embodiment the therapeutic agent is an inhibitor that interactsdirectly with, e.g., binds, either the mutant IDH, e.g., IDH1 or IDH2,protein or interacts directly with, e.g., binds, the mutant IDH mRNA,e.g., IDH1 or IDH2 mRNA.

In an embodiment the therapeutic agent is administered.

In an embodiment the treatment: inhibits, e.g., specifically, aneoactivity of IDH1 or IDH2 (e.g., a neoactivity of IDH1), e.g., aneoactivity described herein; or inhibits both the wildtype and activityand a neoactivity of IDH1 or IDH2 (e.g., a neoactivity of IDH1), e.g., aneoactivity described herein In an embodiment, the subject issubsequently evaluated or monitored by a method described herein, e.g.,the analysis of the presence, distribution, or level of an alpha hydroxyneoactivity product, e.g., 2HG, e.g., R-2HG, e.g., to evaluate responseto the treatment or progression of disease.

In an embodiment the treatment is selected on the basis that it:inhibits, e.g., specifically, a neoactivity of IDH1 or IDH2 (e.g., aneoactivity of IDH1), e.g., alpha hydroxy neoactivity, e.g., 2HGneoactivity; or inhibits both the wildtype and activity and aneoactivity of IDH1 or IDH2 (e.g., a neoactivity of IDH1), e.g., aneoactivity described herein.

In an embodiment, the method comprises determining the possibility of amutation other than a mutation in IDH1 or in IDH2. In embodiments arelatively high level of 2HG, e.g., R-2HG is indicative of anothermutation.

In an embodiment, which embodiment includes selecting or administering atreatment for the subject, the subject:

has not yet been treated for the subject the cell proliferation-relateddisorder and the selected or administered treatment is the initial orfirst line treatment;

has already been treated for the cell proliferation-related and theselected or administered treatment results in an alteration of theexisting treatment;

has already been treated for the cell proliferation-related, and theselected treatment results in continuation of the existing treatment; or

has already been treated for the cell proliferation-related disorder andthe selected or administered treatment is different, e.g., as comparedto what was administered prior to the evaluation or to what would beadministered in the absence of elevated levels of an alpha hydroxyneoactivity product, e.g., 2HG, e.g., R-2HG.

In an embodiment, which embodiment includes selecting or administering atreatment for the subject, the selected or administered treatment cancomprise:

a treatment which includes administration of a therapeutic agent atdifferent, e.g., a greater (or lesser) dosage (e.g., different ascompared to what was administered prior to the evaluation or to whatwould be administered in the absence of elevated levels of an alphahydroxy neoactivity product, e.g., 2HG, e.g., R-2HG);

a treatment which includes administration of a therapeutic agent at adifferent frequency, e.g., more or less frequently, or not at all (e.g.,different as compared to what was administered prior to the evaluationor to what would be administered in the absence of elevated levels of analpha hydroxy neoactivity product, e.g., 2HG, e.g., R-2HG); or

a treatment which includes administration of a therapeutic agent in adifferent therapeutic setting (e.g., adding or deleting a secondtreatment from the treatment regimen) (e.g., different as compared towhat was administered prior to the evaluation or to what would beadministered in the absence of elevated levels of an alpha hydroxyneoactivity product, e.g., 2HG, e.g., R-2HG).

Methods of evaluating a subject described herein can comprise evaluatinga neoactivity genotype or phenotype. Methods of obtaining and analyzingsamples, and the in vivo analysis in subjects, described elsewhereherein, e.g., in the section entitled, “Methods of evaluating samplesand/or subjects,” can be combined with this method.

In an embodiment the method comprises:

subjecting the subject (e.g., a subject not having 2-hydroxyglutaricaciduria) to imaging and/or spectroscopic analysis, e.g., magneticresonance-based analysis, e.g., MRI and/or MRS e.g., imaging analysis,to provide a determination of the presence, distribution, or level of analpha hydroxy neoactivity product, e.g., 2HG, e.g., R-2HG, e.g., asassociated with a tumor, e.g., a glioma, in the subject;

optionally storing a parameter related to the determination, e.g., theimage or a value related to the image from the imaging analysis, in atangible medium; and

responsive to the determination, performing one or more of: correlatingthe determination with outcome or with a prognosis; providing anindication of outcome or prognosis; providing a value for an analysis onwhich the evaluation is based, e.g., the presence, distribution, orlevel of an alpha hydroxy neoactivity product, e.g., 2HG, e.g., R-2HG;providing a recommendation for treatment of the subject; selecting acourse of treatment for the subject, e.g., a course of treatmentdescribed herein, e.g., selecting a course of treatment that includesinhibiting a neoactivity of a mutant IDH, e.g., IDH1 or IDH2, allele,e.g., a neoactivity described herein; administering a course oftreatment to the subject, e.g., a course of treatment described herein,e.g., a course of treatment that includes inhibiting a neoactivity of amutant IDH, e.g., IDH1 or IDH2, allele, e.g., a neoactivity describedherein; and memorializing memorializing a result of the method or ameasurement made in the course of the method, e.g., one or more of theabove and/or transmitting memorialization of one or more of the above toa party, e.g., the subject, a healthcare provider, or an entity thatpays for the subject's treatment, e.g., a government, insurance company,or other third party payer.

In an embodiment the method comprises confirming or determining, e.g.,by direct examination or evaluation of the subject, or sample e.g.,tissue or bodily fluid (e.g., blood (e.g., blood plasma), urine, lymph,or cerebrospinal fluid) therefrom, (e.g., by DNA sequencing or immunoanalysis or evaluation of the presence, distribution or level of analpha hydroxy neoactivity product, e.g., 2HG, e.g., R-2HG), or receivingsuch information about the subject, that the subject has a cancercharacterized by an IDH, e.g., IDH1 or IDH2, allele described herein,e.g., an IDH1 allele having His, Ser, Cys, Gly, Val, Pro or Leu atresidue 132 (SEQ ID NO:8), in specific embodiments, an IDH1 allelehaving His, Ser, Cys, Gly, Val, or Leu at residue 132 or an IDH1 allelehaving His or Cys at residue 132; or an IDH2 allele having Lys, Gly,Met, Trp, Thr, or Ser at residue 172 (SEQ ID NO:10).

In an embodiment, prior to or after treatment, the method includesevaluating the growth, size, weight, invasiveness, stage or otherphenotype of the cell proliferation-related disorder.

In an embodiment the cell proliferation-related disorder is a tumor ofthe CNS, e.g., a glioma, a leukemia, e.g., AML or ALL, e.g., B-ALL orT-ALL, prostate cancer, or myelodysplasia or myelodysplastic syndromeand the evaluation is a or b. In an embodiment the method comprisesevaluating a sample, e.g., a sample described herein, e.g., a tissue,e.g., a cancer sample, or a bodily fluid, e.g., serum or blood, forincreased alpha neoactivity product, e.g., 2HG, e.g., R-2HG.

In an embodiment, a subject is subjected to MRS and the evaluationcomprises evaluating the presence or elevated amount of a peakcorrelated to or corresponding to 2HG, e.g., R-2HG, as determined bymagnetic resonance. For example, a subject can be analyzed for thepresence and/or strength of a signal at about 2.5 ppm to determine thepresence and/or amount of 2HG, e.g., R-2HG in the subject.

In an embodiment the method comprises obtaining a sample from thesubject and analyzing the sample, or analyzing the subject, e.g., byimaging the subject and optionally forming a representation of the imageon a computer.

In an embodiment the results of the analysis is compared to a reference.

In an embodiment a value for a parameter correlated to the presence,distribution, or level, e.g., of 2HG, e.g., R-2HG, is determined. It canbe compared with a reference value, e.g., the value for a referencesubject not having abnormal presence, level, or distribution, e.g., areference subject cell not having a mutation in IDH, e.g., IDH1 or IDH2,having a neoactivity described herein.

In an embodiment the method comprises determining if an IDH, e.g., IDH1or IDH2, mutant allele that is associated with 2HG neoactivity ispresent. E.g., in the case of IDH1, the presence of a mutation atresidue 132 associated with 2HG neoactivity can be determined. In thecase of IDH2, the presence of a mutation at residue 172 associated with2HG neoactivity can be determined. The determination can comprisesequencing a nucleic acid, e.g., genomic DNA or cDNA, from an affectedcell, which encodes the relevant amino acid(s). The mutation can be adeletion, insertion, rearrangement, or substitution. The mutation caninvolve a single nucleotide, e.g., a single substitution, or more thanone nucleotide, e.g., a deletion of more than one nucleotides.

In an embodiment the method comprises determining the sequence atposition 394 or 395 of the IDH1 gene, or determining the identity ofamino acid residue 132 (SEQ ID NO:8) in the IDH1 gene in a cellcharacterized by the cell proliferation related disorder.

In an embodiment the method comprises determining the amino acidsequence, e.g., by DNA sequencing, at position 172 of the IDH2 gene in acell characterized by the cell proliferation related disorder.

In an embodiment a product of the neoactivity is 2-HG, e.g., R-2HG,which acts as a metabolite. In another embodiment a product of theneoactivity is 2HG, e.g., R-2HG, which acts as a toxin, e.g., acarcinogen.

In an embodiment the disorder is other than a solid tumor. In anembodiment the disorder is a tumor that, at the time of diagnosis ortreatment, does not have a necrotic portion. In an embodiment thedisorder is a tumor in which at least 30, 40, 50, 60, 70, 80 or 90% ofthe tumor cells carry an IHD, e.g., IDH1 or IDH2, mutation having 2HGneoactivity, at the time of diagnosis or treatment.

In an embodiment the cell proliferation-related disorder is a cancer,e.g., a cancer described herein, characterized by an IDH1 somatic mutanthaving alpha hydroxy neoactivity, e.g., 2HG neoactivity, e.g., a mutantdescribed herein. In an embodiment the tumor is characterized byincreased levels of an alpha hydroxy neoactivity product, 2HG, e.g.,R-2HG, as compared to non-diseased cells of the same type.

In an embodiment the method comprises selecting a subject having aglioma, on the basis of the cancer being characterized by increasedlevels of an alpha hydroxy neoactivity, product, e.g., 2HG, e.g., R-2HG.

In an embodiment the cell proliferation-related disorder is a tumor ofthe CNS, e.g., a glioma, e.g., wherein the tumor is characterized by anIDH1 somatic mutant having alpha hydroxy neoactivity, e.g., 2HGneoactivity, e.g., a mutant described herein. Gliomas include astrocytictumors, oligodendroglial tumors, oligoastrocytic tumors, anaplasticastrocytomas, and glioblastomas. In an embodiment the tumor ischaracterized by increased levels of an alpha hydroxy neoactivityproduct, e.g., 2HG, e.g., R-2HG, as compared to non-diseased cells ofthe same type. E.g., in an embodiment, the IDH1 allele encodes an IDH1having other than an Arg at residue 132. E.g., the allele encodes His,Ser, Cys, Gly, Val, Pro or Leu, or any residue described in Yan et al.,at residue 132, according to the sequence of SEQ ID NO:8 (see also FIG.21). In an embodiment the allele encodes an IDH1 having His at residue132. In an embodiment the allele encodes an IDH1 having Ser at residue132.

In an embodiment the IDH1 allele has an A (or any other nucleotide otherthan C) at nucleotide position 394, or an A (or any other nucleotideother than G) at nucleotide position 395. In an embodiment the allele isa C394A, a C394G, a C394T, a G395C, a G395T or a G395A mutation,specifically C394A or a G395A mutation according to the sequence of SEQID NO:5.

In an embodiment the method comprises selecting a subject having aglioma, wherein the cancer is characterized by having an IDH1 alleledescribed herein, e.g., an IDH1 allele having His, Ser, Cys, Gly, Val,Pro or Leu at residue 132 (SEQ ID NO:8) (e.g., His, Ser, Cys, Gly, Val,or Leu; or His or Cys).

In an embodiment the method comprises selecting a subject having aglioma, on the basis of the cancer being characterized by an IDH1 alleledescribed herein, e.g., an IDH1 allele having His, Ser, Cys, Gly, Val,Pro or Leu at residue 132 (SEQ ID NO:8) (e.g., His, Ser, Cys, Gly, Val,or Leu; or His or Cys).

In an embodiment the method comprises selecting a subject having aglioma, on the basis of the cancer being characterized by increasedlevels of an alpha hydroxy neoactivity, product, e.g., 2HG, e.g., R-2HG.

In an embodiment, the cell proliferation disorder is fibrosarcoma orparaganglioma wherein the cancer is characterized by having an IDH1allele described herein, e.g., an IDH1 allele having Cys at residue 132(SEQ ID NO:8).

In an embodiment, the cell proliferation disorder is fibrosarcoma orparaganglioma wherein the cancer is characterized by an IDH1 alleledescribed herein, e.g., an IDH1 allele having Cys at residue 132 (SEQ IDNO:8).

In an embodiment, the cell proliferation disorder is fibrosarcoma orparaganglioma wherein the cancer is characterized by increased levels ofan alpha hydroxy neoactivity, product, e.g., 2HG, e.g., R-2HG.

In an embodiment the cell proliferation-related disorder is localized ormetastatic prostate cancer, e.g., prostate adenocarcinoma, e.g., whereinthe cancer is characterized by an IDH1 somatic mutant having alphahydroxy neoactivity, e.g., 2HG neoactivity, e.g., a mutant describedherein. In an embodiment the cancer is characterized by increased levelsof an alpha hydroxy neoactivity product, e.g., 2HG, e.g., R-2HG, ascompared to non-diseased cells of the same type.

E.g., in an embodiment, the IDH1 allele encodes an IDH1 having otherthan an Arg at residue 132. E.g., the allele encodes His, Ser, Cys, Gly,Val, Pro or Leu, or any residue described in Kang et al, 2009, Int. J.Cancer, 125: 353-355 at residue 132, according to the sequence of SEQ IDNO:8 (see also FIG. 21) (e.g., His, Ser, Cys, Gly, Val, or Leu). In anembodiment the allele encodes an IDH1 having His or Cys at residue 132.

In an embodiment the IDH1 allele has a T (or any other nucleotide otherthan C) at nucleotide position 394, or an A (or any other nucleotideother than G) at nucleotide position 395. In an embodiment the allele isa C394T or a G395A mutation according to the sequence of SEQ ID NO:5.

In an embodiment the method comprises selecting a subject havingprostate cancer, e.g., prostate adenocarcinoma, wherein the cancer ischaracterized by an IDH1 allele described herein, e.g., an IDH1 allelehaving His or Cys at residue 132 (SEQ ID NO:8).

In an embodiment the method comprises selecting a subject havingprostate cancer, e.g., prostate adenocarcinoma, on the basis of thecancer being characterized by an IDH1 allele described herein, e.g., anIDH1 allele having His or Cys at residue 132 (SEQ ID NO:8).

In an embodiment the method comprises selecting a subject havingprostate cancer, on the basis of the cancer being characterized byincreased levels of an alpha hydroxy neoactivity product, e.g., 2HG,e.g., R-2HG.

In an embodiment the cell proliferation-related disorder is ahematological cancer, e.g., a leukemia, e.g., AML, or ALL, wherein thehematological cancer is characterized by an IDH1 somatic mutant havingalpha hydroxy neoactivity, e.g., 2HG neoactivity, e.g., a mutantdescribed herein. In an embodiment the cancer is characterized byincreased levels of an alpha hydroxy neoactivity product, e.g., 2HG,e.g., R-2HG, as compared to non-diseased cells of the same type. In anembodiment the method comprises evaluating a serum or blood sample forincreased alpha neoactivity product, e.g., 2HG, e.g., R-2HG.

In an embodiment the cell proliferation-related disorder is acutelymphoblastic leukemia (e.g., an adult or pediatric form), e.g., whereinthe acute lymphoblastic leukemia (sometimes referred to herein as ALL)is characterized by an IDH1 somatic mutant having alpha hydroxyneoactivity, e.g., 2HG neoactivity, e.g., a mutant described herein. TheALL can be, e.g., B-ALL or T-ALL. In an embodiment the cancer ischaracterized by increased levels of 2 an alpha hydroxy neoactivityproduct, e.g., HG, e.g., R-2HG, as compared to non-diseased cells of thesame type. E.g., in an embodiment, the IDH1 allele is an IDH1 havingother than an Arg at residue 132 (SEQ ID NO:8). E.g., the allele encodesHis, Ser, Cys, Gly, Val, Pro or Leu, or any residue described in Kang etal., at residue 132, according to the sequence of SEQ ID NO:8 (see alsoFIG. 21) (e.g., His, Ser, Cys, Gly, Val, or Leu). In an embodiment theallele encodes an IDH1 having Cys at residue 132.

In an embodiment the IDH1 allele has a T (or any other nucleotide otherthan C) at nucleotide position 394. In an embodiment the allele is aC394T mutation according to the sequence of SEQ ID NO:5.

In an embodiment the method comprises selecting a subject having ALL,e.g., B-ALL or T-ALL, characterized by an IDH1 allele described herein,e.g., an IDH1 allele having Cys at residue 132 according to the sequenceof SEQ ID NO:8.

In an embodiment the method comprises selecting a subject ALL, e.g.,B-ALL or T-ALL, on the basis of cancer being characterized by having anIDH1 allele described herein, e.g., an IDH1 allele having Cys at residue132 (SEQ ID NO:8).

In an embodiment the method comprises selecting a subject having ALL,e.g., B-ALL or T-ALL, on the basis of the cancer being characterized byincreased levels of an alpha hydroxy neoactivity product, e.g., 2HG,e.g., R-2HG.

In an embodiment the cell proliferation-related disorder is acutemyelogenous leukemia (e.g., an adult or pediatric form), e.g., whereinthe acute myelogenous leukemia (sometimes referred to herein as AML) ischaracterized by an IDH1 somatic mutant having alpha hydroxyneoactivity, e.g., 2HG neoactivity, e.g., a mutant described herein. Inan embodiment the cancer is characterized by increased levels of analpha hydroxy neoactivity product, e.g., 2HG, e.g., R-2HG, as comparedto non-diseased cells of the same type. E.g., in an embodiment, the IDH1allele is an IDH1 having other than an Arg at residue 132 (SEQ ID NO:8).E.g., the allele encodes His, Ser, Cys, Gly, Val, Pro or Leu, or anyresidue described in Kang et al., at residue 132, according to thesequence of SEQ ID NO:8 (see also FIG. 21) (e.g., His, Ser, Cys, Gly,Val or Leu). In an embodiment the allele encodes an IDH1 having Cys, Hisor Gly at residue 132, specifically, Cys.

In an embodiment the IDH1 allele has a T (or any other nucleotide otherthan C) at nucleotide position 394. In an embodiment the allele is aC394T mutation according to the sequence of SEQ ID NO:5.

In an embodiment the method comprises selecting a subject having acutemyelogenous lymphoplastic leukemia (AML) characterized by an IDH1 alleledescribed herein, e.g., an IDH1 allele having Cys, His or Gly at residue132 according to the sequence of SEQ ID NO:8, specifically, Cys.

In an embodiment the method comprises selecting a subject having acutemyelogenous lymphoplastic leukemia (AML) on the basis of cancer beingcharacterized by having an IDH1 allele described herein, e.g., an IDH1allele having Cys, His or Gly at residue 132 (SEQ ID NO:8),specifically, Cys.

In an embodiment the method comprises selecting a subject having acutemyelogenous lymphoplastic leukemia (AML), on the basis of the cancerbeing characterized by increased levels of an alpha hydroxy neoactivityproduct, e.g., 2HG, e.g., R-2HG. In an embodiment the method comprisesevaluating a serum or blood sample for increased alpha neoactivityproduct, e.g., 2HG, e.g., R-2HG.

In an embodiment the method further comprises evaluating the subject forthe presence of a mutation in the NRAS or NPMc gene.

In an embodiment the cell proliferation-related disorder ismyelodysplasia or myelodysplastic syndrome, e.g., wherein themyelodysplasia or myelodysplastic syndrome is characterized by having anIDH1 somatic mutant having alpha hydroxy neoactivity, e.g., 2HGneoactivity, e.g., a mutant described herein. In an embodiment thedisorder is characterized by increased levels of an alpha hydroxyneoactivity product, e.g., 2HG, e.g., R-2HG, as compared to non-diseasedcells of the same type. E.g., in an embodiment, the IDH1 allele is anIDH1 having other than an Arg at residue 132 (SEQ ID NO:8). E.g., theallele encodes His, Ser, Cys, Gly, Val, Pro or Leu, or any residuedescribed in Kang et al., according to the sequence of SEQ ID NO:8 (seealso FIG. 21), specifically, His, Ser, Cys, Gly, Val, or Leu. In anembodiment the allele encodes an IDH1 having Cys at residue 132.

In an embodiment the IDH1 allele has a T (or any other nucleotide otherthan C) at nucleotide position 394. In an embodiment the allele is aC394T mutation according to the sequence of SEQ ID NO:5.

In an embodiment the method comprises selecting a subject havingmyelodysplasia or myelodysplastic syndrome characterized by an IDH1allele described herein, e.g., an IDH1 allele having Cys at residue 132according to the sequence of SEQ ID NO:8.

In an embodiment the method comprises selecting a subject havingmyelodysplasia or myelodysplastic syndrome on the basis of cancer beingcharacterized by having an IDH1 allele described herein, e.g., an IDH1allele having Cys at residue 132 (SEQ ID NO:8).

In an embodiment the method comprises selecting a subject havingmyelodysplasia or myelodysplastic syndrome, on the basis of the cancerbeing characterized by increased levels of an alpha hydroxy neoactivityproduct, e.g., 2HG, e.g., R-2HG. In an embodiment the method comprisesevaluating a serum or blood sample for increased alpha neoactivityproduct, e.g., 2HG, e.g., R-2HG.

In an embodiment the cell proliferation-related disorder is a glioma,characterized by a mutation, or preselected allele, of IDH2 associatedwith an alpha hydroxy neoactivity, e.g., 2HG neoactivity. E.g., in anembodiment, the IDH2 allele encodes an IDH2 having other than an Arg atresidue 172. E.g., the allele encodes Lys, Gly, Met, Trp, Thr, Ser, orany residue described in described in Yan et al., at residue 172,according to the sequence of SEQ ID NO:10 (see also FIG. 22),specifically, Lys, Gly, Met, Trp or Ser. In an embodiment the alleleencodes an IDH2 having Lys at residue 172. In an embodiment the alleleencodes an IDH2 having Met at residue 172.

In an embodiment the method comprises selecting a subject having aglioma, wherein the cancer is characterized by having an IDH2 alleledescribed herein, e.g., an IDH2 allele having Lys, Gly, Met, Trp, Thr,or Ser at residue 172 (SEQ ID NO:10), specifically Lys, Gly, Met, Trp,or Ser; or Lys or Met.

In an embodiment the method comprises selecting a subject having aglioma, on the basis of the cancer being characterized by an IDH2 alleledescribed herein, e.g., an IDH2 allele having Lys, Gly, Met, Trp, Thr,or Ser at residue 172 (SEQ ID NO:10), specifically Lys, Gly, Met, Trp,or Ser; or Lys or Met.

In an embodiment the method comprises selecting a subject having aglioma, on the basis of the cancer being characterized by increasedlevels of an alpha hydroxy neoactivity product, e.g., 2HG, e.g., R-2HG.

In an embodiment the cell proliferation-related disorder is a prostatecancer, e.g., prostate adenocarcinoma, characterized by a mutation, orpreselected allele, of IDH2 associated with an alpha hydroxyneoactivity, e.g., 2HG neoactivity. E.g., in an embodiment, the IDH2allele encodes an IDH2 having other than an Arg at residue 172. E.g.,the allele encodes Lys, Gly, Met, Trp, Thr, Ser, or any residuedescribed in described in Yan et al., at residue 172, according to thesequence of SEQ ID NO:10 (see also FIG. 22), specifically Lys, Gly, Met,Trp, or Ser. In an embodiment the allele encodes an IDH2 having Lys atresidue 172. In an embodiment the allele encodes an IDH2 having Met atresidue 172.

In an embodiment the method comprises selecting a subject having aprostate cancer, e.g., prostate adenocarcinoma, wherein the cancer ischaracterized by having an IDH2 allele described herein, e.g., an IDH2allele having Lys or Met at residue 172 (SEQ ID NO:10).

In an embodiment the method comprises selecting a subject having aprostate cancer, e.g., prostate adenocarcinoma, on the basis of thecancer being characterized by an IDH2 allele described herein, e.g., anIDH2 allele having Lys or Met at residue 172 (SEQ ID NO:10).

In an embodiment the method comprises selecting a subject having aprostate cancer, e.g., prostate adenocarcinoma, on the basis of thecancer being characterized by increased levels of an alpha hydroxyneoactivity product, e.g., 2HG, e.g., R-2HG.

In an embodiment the cell proliferation-related disorder is ALL, e.g.,B-ALL or T-ALL, characterized by a mutation, or preselected allele, ofIDH2 associated with an alpha hydroxy neoactivity, e.g., 2HGneoactivity. E.g., in an embodiment, the IDH2 allele encodes an IDH2having other than an Arg at residue 172. E.g., the allele encodes Lys,Gly, Met, Trp, Thr, Ser, or any residue described in described in Yan etal., at residue 172, according to the sequence of SEQ ID NO:10 (see alsoFIG. 22), specifically Lys, Gly, Met, Trp, or Ser. In an embodiment theallele encodes an IDH2 having Lys at residue 172. In an embodiment theallele encodes an IDH2 having Met at residue 172.

In an embodiment the method comprises selecting a subject having ALL,e.g., B-ALL or T-ALL, wherein the cancer is characterized by having anIDH2 allele described herein, e.g., an IDH2 allele having Lys or Met atresidue 172 (SEQ ID NO:10).

In an embodiment the method comprises selecting a subject having ALL,e.g., B-ALL or T-ALL, on the basis of the cancer being characterized byan IDH2 allele described herein, e.g., an IDH2 allele having Lys or Metat residue 172 (SEQ ID NO:10).

In an embodiment the method comprises selecting a subject having ALL,e.g., B-ALL or T-ALL, on the basis of the cancer being characterized byincreased levels of an alpha hydroxy neoactivity product, e.g., 2HG,e.g., R-2HG. In an embodiment the method comprises evaluating a serum orblood sample for increased alpha neoactivity product, e.g., 2HG, e.g.,R-2HG.

In an embodiment the cell proliferation-related disorder is AML,characterized by a mutation, or preselected allele, of IDH2 associatedwith an alpha hydroxy neoactivity, e.g., 2HG neoactivity. E.g., in anembodiment, the IDH2 allele encodes an IDH2 having other than an Arg atresidue 172. E.g., the allele encodes Lys, Gly, Met, Trp, Thr, Ser, orany residue described in described in Yan et al., at residue 172,according to the sequence of SEQ ID NO:10 (see also FIG. 22),specifically Lys, Gly, Met, Trp, or Ser. In an embodiment the alleleencodes an IDH2 having Lys at residue 172. In an embodiment the alleleencodes an IDH2 having Met at residue 172.

In an embodiment the method comprises selecting a subject having AML,wherein the cancer is characterized by having an IDH2 allele describedherein, e.g., an IDH2 allele having Lys or Met at residue 172 (SEQ IDNO:10).

In an embodiment the method comprises selecting a subject having AML, onthe basis of the cancer being characterized by an IDH2 allele describedherein, e.g., an IDH2 allele having Lys or Met at residue 172 (SEQ IDNO:10).

In an embodiment the method comprises selecting a subject having AML, onthe basis of the cancer being characterized by increased levels of analpha hydroxy neoactivity product, e.g., 2HG, e.g., R-2HG. In anembodiment the method comprises evaluating a serum or blood sample forincreased alpha neoactivity product, e.g., 2HG, e.g., R-2HG.

In an embodiment the cell proliferation-related disorder ismyelodysplasia or myelodysplastic syndrome, characterized by a mutation,or preselected allele, of IDH2. E.g., in an embodiment, the IDH2 alleleencodes an IDH2 having other than an Arg at residue 172. E.g., theallele encodes Lys, Gly, Met, Trp, Thr, Ser, or any residue described indescribed in Yan et al., at residue 172, according to the sequence ofSEQ ID NO:10 (see also FIG. 22), specifically Lys, Gly, Met, Trp, orSer. In an embodiment the allele encodes an IDH2 having Lys at residue172. In an embodiment the allele encodes an IDH2 having Met at residue172.

In an embodiment the method comprises selecting a subject havingmyelodysplasia or myelodysplastic syndrome, wherein the cancer ischaracterized by having an IDH2 allele described herein, e.g., an IDH2allele having Lys or Met at residue 172 (SEQ ID NO:10).

In an embodiment the method comprises selecting a subject havingmyelodysplasia or myelodysplastic syndrome, on the basis of the cancerbeing characterized by an IDH2 allele described herein, e.g., an IDH2allele having Lys or Met at residue 172 (SEQ ID NO:10).

In an embodiment the method comprises selecting a subject havingmyelodysplasia or myelodysplastic syndrome, on the basis of the cancerbeing characterized by increased levels of an alpha hydroxy neoactivityproduct, e.g., 2HG, e.g., R-2HG. In an embodiment the method comprisesevaluating a serum or blood sample for increased alpha neoactivityproduct, e.g., 2HG, e.g., R-2HG.

In another aspect the invention features a pharmaceutical composition ofan inhibitor (e.g., a small molecule or a nucleic acid-based inhibitor)described herein.

In an embodiment a mutant protein specific reagent, e.g., an antibodythat specifically binds an IDH mutant protein, e.g., an antibody thatspecifically binds an IDH1-R132H mutant protein, can be used to detectneoactive mutant enzyme see, for example, that described by Y. Kato etal., “A monoclonal antibody IMab-1 specifically recognizes IDH1^(R132H),the most common glioma-derived mutation: (Kato, Biochem. Biophys. Res.Commun. (2009), which is hereby incorporated by reference in itsentirety.

In another aspect, the invention features, a method of evaluating acandidate compound, e.g., for the ability to inhibit a neoactivity of amutant enzyme, e.g., for use as an anti-proliferative or anti-canceragent. In an embodiment the mutant enzyme is an IDH, e.g., an IDH1 orIDH2 mutant, e.g., a mutant described herein. In an embodiment theneaoctivity is alpha hydroxy neoactivity, e.g., 2HG neoactivity. Themethod comprises:

optionally supplying the candidate compound;

contacting the candidate compound with a mutant enzyme having aneoactivity, or with another enzyme, a referred to herein as a proxyenzyme, having an activity, referred to herein as a proxy activity,which is the same as the neoactivity (or with a cell or cell lysatecomprising the same); and

evaluating the ability of the candidate compound to modulate, e.g.,inhibit or promote, the neoactivity or the proxy activity,

thereby evaluating the candidate compound.

In an embodiment the mutant enzyme is a mutant IDH1, e.g., an IDH1mutant described herein, and the neoactivity is an alpha hydroxyneoactivity, e.g., 2HG neoactivity. Mutations associated with 2HGneoactivity in IDH1 include mutations at residue 132, e.g., R132H,R132C, R132S, R132G, R132L, or R132V, more specifically, R132H or R132C.

In an embodiment the mutant enzyme is a mutant IDH2, e.g., an IDH2mutant described herein, and the neoactivity is an alpha hydroxyneoactivity, e.g., 2HG neoactivity. Mutations associated with 2HGneoactivity in IDH2 include mutations at residue 172, e.g., R172K,R172M, R172S, R172G, or R172W.

In an embodiment the method includes evaluating the ability of thecandidate compound to inhibit the neoactivity or the proxy activity.

In an embodiment the method further comprises evaluating the ability ofthe candidate compound to inhibit the forward reaction of non-mutant orwild type enzyme activity, e.g., in the case of IDH, e.g., IDH1 or IDH2,the conversion of isocitrate to α-ketoglutarate (or an intermediatethereof, including the reduced hydroxyl intermediate).

In an embodiment, the contacting step comprises contacting the candidatecompound with a cell, or a cell lysate thereof, wherein the cellcomprises a mutant enzyme having the neoactivity or an enzyme having theactivity.

In an embodiment, the cell comprises a mutation, or preselected allele,of a mutant IDH1 gene. E.g., in an embodiment, the IDH1 allele encodesan IDH1 having other than an Arg at residue 132. E.g., the allele canencode His, Ser, Cys, Gly, Val, Pro or Leu, or any other residuedescribed in Yan et al., at residue 132, according to the sequence ofSEQ ID NO:8 (see also FIG. 21), specifically His, Ser, Cys, Gly, Val, orLeu.

In an embodiment the allele encodes an IDH1 having His at residue 132.

In an embodiment the allele encodes an IDH1 having Ser at residue 132.

In an embodiment the allele is an Arg132His mutation, or an Arg132Sermutation, according to the sequence of SEQ ID NO:8 (see FIGS. 2 and 21).

In an embodiment, the cell comprises a mutation, or preselected allele,of a mutant IDH2 gene. E.g., in an embodiment, the IDH2 allele encodesan IDH2 having other than an Arg at residue 172. E.g., the alleleencodes Lys, Gly, Met, Trp, Thr, Ser, or any residue described indescribed in Yan et al., at residue 172, according to the sequence ofSEQ ID NO:10 (see also FIG. 22), specifically, Lys, Gly, Met, Trp, orSer. In an embodiment the allele encodes an IDH2 having Lys at residue172. In an embodiment the allele encodes an IDH2 having Met at residue172.

In an embodiment, the cell includes a heterologous copy of a mutant IDHgene, e.g., a mutant IDH1 or IDH2 gene. (Heterologous copy refers to acopy introduced or formed by a genetic engineering manipulation.)

In an embodiment, the cell is transfected (e.g., transiently or stablytransfected) or transduced (e.g., transiently or stably transduced) witha nucleic acid sequence encoding an IDH, e.g., IDH1 or IDH2, describedherein, e.g., an IDH1 having other than an Arg at residue 132. In anembodiment, the IDH, e.g., IDH1 or IDH2, is epitope-tagged, e.g.,myc-tagged.

In an embodiment, the cell, e.g., a cancer cell, is non-mutant or wildtype for the IDH, e.g., IDH1 or IDH2, allele. The cell can include aheterologous IDH1 or IDH2 mutant.

In an embodiment, the cell is a cultured cell, e.g., a primary cell, asecondary cell, or a cell line. In an embodiment, the cell is a cancercell, e.g., a glioma cell (e.g., a glioblastoma cell), a prostate cancercell, a leukemia cell (e.g., an ALL, e.g., B-ALL or T-ALL, cell or AMLcell) or a cell characterized by myelodysplasia or myelodysplasticsyndrome. In embodiment, the cell is a 293T cell, a U87MG cell, or anLN-18 cell (e.g., ATCC HTB-14 or CRL-2610).

In an embodiment, the cell is from a subject, e.g., a subject havingcancer, e.g., a cancer characterized by an IDH, e.g., IDH1 or IDH2,allele described herein, e.g., an IDH1 allele having His, Ser, Cys, Gly,Val, Pro or Leu at residue 132 (SEQ ID NO:8); specifically His or Cys;or an IDH2 allele having Lys, Gly, Met, Trp, Thr, or Ser at residue 172(SEQ ID NO:10), specifically Lys, Gly, Met, Trp, or Ser.

In an embodiment, the evaluating step comprises evaluating the presenceand/or amount of an alpha hydroxy neoactivity product, e.g., 2HG, e.g.,R-2HG, e.g., in the cell lysate or culture medium, e.g., by LC-MS.

In an embodiment, the evaluating step comprises evaluating the presenceand/or amount of an alpha hydroxy neoactivity, e.g., 2HG neoactivity, inthe cell lysate or culture medium.

In an embodiment, the method further comprises evaluating thepresence/amount one or more of TCA metabolite(s), e.g., citrate, α-KG,succinate, fumarate, and/or malate, e.g., by LC-MS, e.g., as a control.

In an embodiment, the method further comprises evaluating the oxidationstate of NADPH, e.g., the absorbance at 340 nm, e.g., byspectrophotometer.

In an embodiment, the method further comprises evaluating the ability ofthe candidate compound to inhibit a second enzymatic activity, e.g., theforward reaction of non-mutant or wild type enzyme activity, e.g., inthe case of IDH1 or IDH2 (e.g., IDH1), the conversion of isocitrate toα-ketoglutarate (or an intermediate thereof, including the reducedhydroxyl intermediate).

In an embodiment, the candidate compound is a small molecule, apolypeptide, peptide, a carbohydrate based molecule, or an aptamer(e.g., a nucleic acid aptamer, or a peptide aptamer). The method can beused broadly and can, e.g., be used as one or more of a primary screen,to confirm candidates produced by this or other methods or screens, orgenerally to guide drug discovery or drug candidate optimization.

In an embodiment, the method comprises evaluating, e.g., confirming, theability of a candidate compound (e.g., a candidate compound which meetsa predetermined level of inhibition in the evaluating step) to inhibitthe neoactivity or proxy activity in a second assay.

In an embodiment, the second assay comprises repeating one or more ofthe contacting and/or evaluating step(s) of the basic method.

In another embodiment, the second assay is different from the first.E.g., where the first assay can use a cell or cell lysate or othernon-whole animal model the second assay can use an animal model, e.g., atumor transplant model, e.g., a mouse having an IDH, e.g., IDH1 or IDH2,mutant cell or tumor transplanted in it. E.g., a U87 cell, or glioma,e.g., glioblastoma, cell, harboring a transfected IDH, e.g., IDH1 orIDH2, neoactive mutant can be implanted as a xenograft and used in anassay. Primary human glioma or AML tumor cells can be grafted into miceto allow propogation of the tumor and used in an assay. A geneticallyengineered mouse model (GEMM) harboring an IDH1 or IDH2 mutation and/orother mutation, e.g., a p53 null mutation, can also be used in an assay.

In an embodiment the method comprises:

optionally supplying the candidate compound;

contacting the candidate compound with a cell comprising a nucleic acidsequence, e.g., a heterologous sequence, encoding an IDH1 having otherthan an Arg at residue 132 (e.g., IDH1R132H) or an IDH2 having otherthan an Arg at residue 172 (specifically an IDH1 having other than anArg at residue 132); and

evaluating the presence and/or amount of an alpha hydroxy neoactivityproduct, e.g., 2HG, e.g., R-2HG, in the cell lysate or culture medium,by LC-MS, thereby evaluating the compound.

In an embodiment the result of the evaluation is compared with areference, e.g., the level of product, e.g., an alpha hydroxyneoactivity product, e.g., 2HG. e.g., R-2HG, in a control cell, e.g., acell having inserted therein a wild type or non-mutant copy of IDH1 orIDH2 (e.g., IDH1).

In another aspect, the invention features, a method of evaluating acandidate compound, e.g., for the ability to inhibit an RNA encoding amutant enzyme having a neoactivity, e.g., for use as ananti-proliferative or anti-cancer agent. In an embodiment the mutantenzyme is an IDH, e.g., an IDH1 or IDH2 mutant, e.g., a mutant describedherein. In an embodiment the neaoctivity is alpha hydroxy neoactivity,e.g., 2HG neoactivity. The method comprises:

optionally supplying the candidate compound, e.g., a nucleic acid basedinhibitor (e.g., a dsRNA (e.g., siRNA or shRNA), an antisense, or amicroRNA);

contacting the candidate compound with an RNA, e.g., an mRNA, whichencodes IDH, e.g., an IDH1 or IDH2, e.g., an RNA that encode mutantenzyme having a neoactivity (or with a cell or cell lysate comprisingthe same); and

evaluating the ability of the candidate compound to inhibit the RNA,thereby evaluating the candidate compound. By inhibit the RNA means,e.g., to cleave or otherwise inactivate the RNA.

In an embodiment the RNA encodes a fusion of all or part of the IDH,e.g., IDH1 or IDH2, wildtype or mutant protein to a second protein,e.g., a reporter protein, e.g., a fluorescent protein, e.g., a green orred fluorescent protein.

In an embodiment the mutant enzyme is a mutant IDH1, e.g., an IDH1mutant described herein, and the neoactivity is an alpha hydroxyneoactivity, e.g., 2HG neoactivity.

In an embodiment the mutant enzyme is a mutant IDH2, e.g., an IDH2mutant described herein, and the neoactivity is an alpha hydroxyneoactivity, e.g., 2HG neoactivity.

In an embodiment, the contacting step comprises contacting the candidatecompound with a cell, or a cell lysate thereof, wherein the cellcomprises RNA encoding IDH, e.g., IDH1 or IDH2, e.g., a mutant IDH,e.g., IDH1 or IDH2, enzyme having the neoactivity.

In an embodiment, the cell comprises a mutation, or preselected allele,of a mutant IDH1 gene. E.g., in an embodiment, the IDH1 allele encodesan IDH1 having other than an Arg at residue 132. E.g., the allele canencode His, Ser, Cys, Gly, Val, Pro or Leu, or any other residuedescribed in Yan et al., at residue 132, according to the sequence ofSEQ ID NO:8 (see also FIG. 21), specifically His, Ser, Cys, Gly, Val, orLeu.

In an embodiment the allele encodes an IDH1 having His at residue 132.

In an embodiment the allele encodes an IDH1 having Ser at residue 132.

In an embodiment the allele is an Arg132His mutation, or an Arg132Sermutation, according to the sequence of SEQ ID NO:8 (see FIGS. 2 and 21).

In an embodiment, the cell comprises a mutation, or preselected allele,of a mutant IDH2 gene. E.g., in an embodiment, the IDH2 allele encodesan IDH2 having other than an Arg at residue 172. E.g., the alleleencodes Lys, Gly, Met, Trp, Thr, Ser, or any residue described indescribed in Yan et al., at residue 172, according to the sequence ofSEQ ID NO:10 (see also FIG. 22), specifically Lys, Gly, Met, Trp or Ser.In an embodiment the allele encodes an IDH2 having Lys at residue 172.In an embodiment the allele encodes an IDH2 having Met at residue 172.

In an embodiment, the cell includes a heterologous copy of a wildtype ormutant IDH gene, e.g., a wildtype or mutant IDH1 or IDH2 gene.(Heterologous copy refers to a copy introduced or formed by a geneticengineering manipulation.) In an embodiment the heterologous genecomprises a fusion to a reporter protein, e.g., a fluorescent protein,e.g., a green or red fluorescent protein.

In an embodiment, the cell is transfected (e.g., transiently or stablytransfected) or transduced (e.g., transiently or stably transduced) witha nucleic acid sequence encoding an IDH, e.g., IDH1 or IDH2, describedherein, e.g., an IDH1 having other than an Arg at residue 132 or an IDH2having other than an Arg at residue 172 (e.g., an IDH1 having other thanan Arg at residue 132). In an embodiment, the IDH, e.g., IDH1 or IDH2,is epitope-tagged, e.g., myc-tagged.

In an embodiment, the cell, e.g., a cancer cell, is non-mutant or wildtype for the IDH, e.g., IDH1 or IDH2, allele. The cell can include aheterologous IDH1 or IDH2 mutant.

In an embodiment, the cell is a cultured cell, e.g., a primary cell, asecondary cell, or a cell line. In an embodiment, the cell is a cancercell, e.g., a glioma cell (e.g., a glioblastoma cell), a prostate cancercell, a leukemia cell (e.g., an ALL, e.g., B-ALL or T-ALL cell or AMLcell) or a cell characterized by myelodysplasia or myelodysplasticsyndrome. In embodiment, the cell is a 293T cell, a U87MG cell, or anLN-18 cell (e.g., ATCC HTB-14 or CRL-2610).

In an embodiment, the cell is from a subject, e.g., a subject havingcancer, e.g., a cancer characterized by an IDH, e.g., IDH1 or IDH2,allele described herein, e.g., an IDH1 allele having His, Ser, Cys, Gly,Val, Pro or Leu at residue 132 (SEQ ID NO:8); specifically His or Cys.In an embodiment, the cancer is characterized by an IDH2 allele havingLys, Gly, Met, Trp, Thr, or Ser at residue 172 (SEQ ID NO:10),specifically Lys, Gly, Met, Trp, or Ser.

In an embodiment, the method comprises a second assay and the secondassay comprises repeating one or more of the contacting and/orevaluating step(s) of the basic method.

In another embodiment, the second assay is different from the first.E.g., where the first assay can use a cell or cell lysate or othernon-whole animal model the second assay can use an animal model

In an embodiment the efficacy of the candidate is evaluated by itseffect on reporter protein activity.

In another aspect, the invention features, a method of evaluating acandidate compound, e.g., for the ability to inhibit transcription of anRNA encoding a mutant enzyme having a neoactivity, e.g., for use as ananti-proliferative or anti-cancer agent. In an embodiment the mutantenzyme is an IDH, e.g., an IDH1 or IDH2 mutant, e.g., a mutant describedherein. In an embodiment the neaoctivity is alpha hydroxy neoactivity,e.g., 2HG neoactivity. The method comprises:

optionally supplying the candidate compound, e.g., a small molecule,polypeptide, peptide, aptomer, a carbohydrate-based molecule or nucleicacid based molecule;

contacting the candidate compound with a system comprising a cell orcell lysate; and

evaluating the ability of the candidate compound to inhibit thetranslation of IDH, e.g., IDH1 or IDH2, RNA, e.g, thereby evaluating thecandidate compound.

In an embodiment the system comprises a fusion gene encoding of all orpart of the IDH, e.g., IDH1 or IDH2, wildtype or mutant protein to asecond protein, e.g., a reporter protein, e.g., a fluorescent protein,e.g., a green or red fluorescent protein.

In an embodiment the mutant enzyme is a mutant IDH1, e.g., an IDH1mutant described herein, and the neoactivity is alpha hydroxyneoactivity, e.g., 2HG neoactivity.

In an embodiment the mutant enzyme is a mutant IDH2, e.g., an IDH2mutant described herein, and the neoactivity is alpha hydroxyneoactivity, e.g., 2HG neoactivity.

In an embodiment, the system includes a heterologous copy of a wildtypeor mutant IDH gene, e.g., a wildtype or mutant IDH1 or IDH2 gene.(Heterologous copy refers to a copy introduced or formed by a geneticengineering manipulation.) In an embodiment the heterologous genecomprises a fusion to a reporter protein, e.g., a fluorescent protein,e.g., a green or red fluorescent protein.

In an embodiment the cell, e.g., a cancer cell, is non-mutant or wildtype for the IDH, e.g., IDH1 or IDH2, allele. The cell can include aheterologous IDH1 or IDH2 mutant.

In an embodiment, the cell is a cultured cell, e.g., a primary cell, asecondary cell, or a cell line. In an embodiment, the cell is a cancercell, e.g., a glioma cell (e.g., a glioblastoma cell), a prostate cancercell, a leukemia cell (e.g., an ALL, e.g., B-ALL or T-ALL, cell or AMLcell) or a cell characterized by myelodysplasia or myelodysplasticsyndrome. In embodiment, the cell is a 293T cell, a U87MG cell, or anLN-18 cell (e.g., ATCC HTB-14 or CRL-2610).

In an embodiment, the cell is from a subject, e.g., a subject havingcancer, e.g., a cancer characterized by an IDH, e.g., IDH1 or IDH2,allele described herein, e.g., an IDH1 allele having His, Ser, Cys, Gly,Val, Pro or Leu at residue 132 (SEQ ID NO:8); specifically His, Ser,Cys, Gly, Val, or Leu. In an embodiment, the cancer is characterized anIDH2 allele having Lys, Gly, Met, Trp, Thr, or Ser at residue 172 (SEQID NO:10).

In an embodiment, the method comprises a second assay and the secondassay comprises repeating the method.

In another embodiment, the second assay is different from the first.E.g., where the first assay can use a cell or cell lysate or othernon-whole animal model the second assay can use an animal model.

In an embodiment the efficacy of the candidate is evaluated by itseffect on reporter protein activity.

In another aspect, the invention features, a method of evaluating acandidate compound, e.g., a therapeutic agent, or inhibitor, describedherein in an animal model. The candidate compound can be, e.g., a smallmolecule, polypeptide, peptide, aptomer, a carbohydrate-based moleculeor nucleic acid based molecule. The method comprises, contacting thecandidate with the animal model and evaluating the animal model.

In an embodiment evaluating comprises;

determining an effect of the compound on the general health of theanimal;

determining an effect of the compound on the weight of the animal;

determining an effect of the compound on liver function, e.g, on a liverenzyme;

determining an effect of the compound on the cardiovascular system ofthe animal;

determining an effect of the compound on neurofunction, e.g., onneuromuscular control or response;

determining an effect of the compound on eating or drinking;

determining the distribution of the compound in the animal;

determining the persistence of the compound in the animal or in a tissueor organ of the animal, e.g., determining plasma half-life; or

determining an effect of the compound on a selected cell in the animal;

determining an effect of the compound on the growth, size, weight,invasiveness or other phenotype of a tumor, e.g., an endogenous tumor ora tumor arising from introduction of cells from the same or a differentspecies.

In an embodiment the animal is a non-human primate, e.g., a cynomolgusmonkey or chimpanzee.

In an embodiment the animal is a rodent, e.g., a rat or mouse.

In an embodiment the animal is a large animal, e.g., a dog or pig, otherthan a non-human primate.

In an embodiment the evaluation is memorialized and optionallytransmitted to another party.

In one aspect, the invention provides, a method of evaluating orprocessing a therapeutic agent, e.g., a therapeutic agent referred toherein, e.g., a therapeutic agent that results in a lowering of thelevel of a product of an IDH, e.g., IDH1 or IDH2, mutant having aneoactivity. In an embodiment the neoactivity is an alpha hydroxyneoactivity, e.g., 2HG neoactivity, and the level of an alpha hydroxyneoactivity product, e.g., 2HG, e.g., R-2HG, is lowered.

The method includes:

providing, e.g., by testing a sample, a value (e.g., a test value) for aparameter related to a property of the therapeutic agent, e.g., theability to inhibit the conversion of alpha ketoglutarate to 2hydroxyglutarate (i.e., 2HG), e.g., R-2 hydroxyglutarate (i.e., R-2HG),and,

optionally, providing a determination of whether the value determinedfor the parameter meets a preselected criterion, e.g., is present, or ispresent within a preselected range,

thereby evaluating or processing the therapeutic agent.

In an embodiment the therapeutic agent is approved for use in humans bya government agency, e.g., the FDA.

In an embodiment the parameter is correlated to the ability to inhibit2HG neoactivity, and, e.g., the therapeutic agent is an inhibitor whichbinds to IDH1 or IDH2 protein and reduces an alpha hydroxy neoactivity,e.g., 2HG neoactivity.

In an embodiment the parameter is correlated to the level of mutant IDH,e.g., IDH1 or IDH2, protein, and, e.g., the therapeutic agent is aninhibitor which reduces the level of IDH1 or IDH2 mutant protein.

In an embodiment the parameter is correlated to the level of an RNA thatencodes a mutant IDH, e.g., IDH1 or IDH2, protein, and, e.g., thetherapeutic agent reduces the level of RNA, e.g., mRNA, that encodesIDH1 or IDH2 mutant protein.

In an embodiment the method includes contacting the therapeutic agentwith a mutant IDH, e.g., IDH1 or IDH2, protein (or corresponding RNA).

In an embodiment, the method includes providing a comparison of thevalue determined for a parameter with a reference value or values, tothereby evaluate the therapeutic agent. In an embodiment, the comparisonincludes determining if a test value determined for the therapeuticagent has a preselected relationship with the reference value, e.g.,determining if it meets the reference value. The value need not be anumerical value but, e.g., can be merely an indication of whether anactivity is present.

In an embodiment the method includes determining if a test value isequal to or greater than a reference value, if it is less than or equalto a reference value, or if it falls within a range (either inclusive orexclusive of one or both endpoints). In an embodiment, the test value,or an indication of whether the preselected criterion is met, can bememorialized, e.g., in a computer readable record.

In an embodiment, a decision or step is taken, e.g., a sample containingthe therapeutic agent, or a batch of the therapeutic agent, isclassified, selected, accepted or discarded, released or withheld,processed into a drug product, shipped, moved to a different location,formulated, labeled, packaged, contacted with, or put into, a container,e.g., a gas or liquid tight container, released into commerce, or soldor offered for sale, or a record made or altered to reflect thedetermination, depending on whether the preselected criterion is met.E.g., based on the result of the determination or whether an activity ispresent, or upon comparison to a reference standard, the batch fromwhich the sample is taken can be processed, e.g., as just described.

The evaluation of the presence or level of activity can show if thetherapeutic agent meets a reference standard.

In an embodiment, methods and compositions disclosed herein are usefulfrom a process standpoint, e.g., to monitor or ensure batch-to-batchconsistency or quality, or to evaluate a sample with regard to areference, e.g., a preselected value.

In an embodiment, the method can be used to determine if a test batch ofa therapeutic agent can be expected to have one or more of theproperties. Such properties can include a property listed on the productinsert of a therapeutic agent, a property appearing in a compendium,e.g., the US Pharmacopea, or a property required by a regulatory agency,e.g., the FDA, for commercial use.

In an embodiment the method includes testing the therapeutic agent forits effect on the wildtype activity of an IDH, e.g., IDH1 or IDH2,protein, and providing a determination of whether the value determinedmeets a preselected criterion, e.g., is present, or is present within apreselected range.

In an embodiment the method includes:

contacting a therapeutic agent that is an inhibitor of IDH1 an alphahydroxy neoactivity, e.g., 2HG neoactivity, with an IDH1 mutant havingan alpha hydroxy neoactivity, e.g., 2HG neoactivity,

determining a value related to the inhibition of an alpha hydroxyneoactivity, e.g., 2HG neoactivity, and

comparing the value determined with a reference value, e.g., a range ofvalues, for the inhibition of an alpha hydroxy neoactivity, e.g., 2HGneoactivity. In an embodiment the reference value is an FDA requiredvalue, e.g., a release criteria.

In an embodiment the method includes:

contacting a therapeutic agent that is an inhibitor of mRNA whichencodes a mutant IDH1 having an alpha hydroxy neoactivity, e.g., 2HGneoactivity, with an mRNA that encodes an IDH1 mutant having an alphahydroxy neoactivity, e.g., 2HG neoactivity,

determining a value related to the inhibition of the mRNA, and,

comparing the value determined with a reference value, e.g., a range ofvalues for inhibition of the mRNA. In an embodiment the reference valueis an FDA required value, e.g., a release criteria.

In one aspect, the invention features a method of evaluating a sample ofa therapeutic agent, e.g., a therapeutic agent referred to herein, thatincludes receiving data with regard to an activity of the therapeuticagent; providing a record which includes said data and optionallyincludes an identifier for a batch of therapeutic agent; submitting saidrecord to a decision-maker, e.g., a government agency, e.g., the FDA;optionally, receiving a communication from said decision maker;optionally, deciding whether to release market the batch of therapeuticagent based on the communication from the decision maker. In oneembodiment, the method further includes releasing, or other wiseprocessing, e.g., as described herein, the sample.

In another aspect, the invention features, a method of selecting apayment class for treatment with a therapeutic agent described herein,e.g., an inhibitor of IDH, e.g., IDH1 or IDH2, neoactivity, for asubject having a cell proliferation-related disorder. The methodincludes:

providing (e.g., receiving) an evaluation of whether the subject ispositive for increased levels of an alpha hydroxy neoactivity product,e.g., 2HG, e.g., R-2HG, or neoactivity, e.g., an alpha hydroxyneoactivity, e.g., 2HG neoactivity, a mutant IDH1 or IDH2 havingneoactivity, e.g., an alpha hydroxy neoactivity, e.g., 2HG neoactivity,(or a corresponding RNA), or a mutant IDH, e.g., IDH1 or IDH2, somaticgene, e.g., a mutant described herein, and

performing at least one of (1) if the subject is positive selecting afirst payment class, and (2) if the subject is a not positive selectinga second payment class.

In an embodiment the selection is memorialized, e.g., in a medicalrecords system.

In an embodiment the method includes evaluation of whether the subjectis positive for increased levels of an alpha hydroxy neoactivityproduct, e.g., 2HG, e.g., R-2HG, or neoactivity, e.g., an alpha hydroxyneoactivity, e.g., 2HG neoactivity.

In an embodiment the method includes requesting the evaluation.

In an embodiment the evaluation is performed on the subject by a methoddescribed herein.

In an embodiment, the method comprises communicating the selection toanother party, e.g., by computer, compact disc, telephone, facsimile,email, or letter.

In an embodiment, the method comprises making or authorizing payment forsaid treatment.

In an embodiment, payment is by a first party to a second party. In someembodiments, the first party is other than the subject. In someembodiments, the first party is selected from a third party payor, aninsurance company, employer, employer sponsored health plan, HMO, orgovernmental entity. In some embodiments, the second party is selectedfrom the subject, a healthcare provider, a treating physician, an HMO, ahospital, a governmental entity, or an entity which sells or suppliesthe drug. In some embodiments, the first party is an insurance companyand the second party is selected from the subject, a healthcareprovider, a treating physician, an HMO, a hospital, a governmentalentity, or an entity which sells or supplies the drug. In someembodiments, the first party is a governmental entity and the secondparty is selected from the subject, a healthcare provider, a treatingphysician, an HMO, a hospital, an insurance company, or an entity whichsells or supplies the drug.

As used herein, a cell proliferation-related disorder is a disordercharacterized by unwanted cell proliferation or by a predisposition tolead to unwanted cell proliferation (sometimes referred to as aprecancerous disorder). Examples of disorders characterized by unwantedcell proliferation include cancers, e.g., tumors of the CNS, e.g., aglioma. Gliomas include astrocytic tumors, oligodendroglial tumors,oligoastrocytic tumors, anaplastic astrocytomas, and glioblastomas.Other examples include hematological cancers, e.g., a leukemia, e.g.,AML (e.g., an adult or pediatric form) or ALL, e.g., B-ALL or T-ALL(e.g., an adult or pediatric form), localized or metastatic prostatecancer, e.g., prostate adenocarcinoma, fibrosarcoma, and paraganglioma;specifically leukemia, e.g., AML (e.g., an adult or pediatric form) orALL, e.g., B-ALL or T-ALL (e.g., an adult or pediatric form), localizedor metastatic prostate cancer, e.g., prostate adenocarcinoma. Examplesof disorders characterized by a predisposition to lead to unwanted cellproliferation include myelodysplasia or myelodysplastic syndrome, whichare a diverse collection of hematological conditions marked byineffective production (or dysplasia) of myeloid blood cells and risk oftransformation to AML.

As used herein, specifically inhibits a neoactivity (and similarlanguage), means the neoactivity of the mutant enzyme is inhibited to asignificantly greater degree than is the wildtype enzyme activity. Byway of example, “specifically inhibits the 2HG neoactivity of mutantIDH1 (or IDH2)” means the 2HG neoactivity is inhibited to asignificantly greater degree than is the forward reaction (theconversion of isocitrate to alpha ketoglutarate) of wildtype IDH1 (orIDH2) activity. In embodiments the neoactivity is inhibited at least 2,5, 10, or 100 fold more than the wildtype activity. In embodiments aninhibitor that is specific for the 2HG neaoctivity of IDH, e.g., IDH1 orIDH2, will also inhibit another dehydrogenase, e.g., malatedehydrogenase. In other embodiments the specific inhibitor does inhibitother dehydrogenases, e.g., malate dehydrogenase.

As used herein, a cell proliferation-related disorder, e.g., a cancer,characterized by a mutation or allele, means a cellproliferation-related disorder having a substantial number of cellswhich carry that mutation or allele. In an embodiment at least 10, 25,50, 75, 90, 95 or 99% of the cell proliferation-related disorder cells,e.g., the cells of a cancer, or a representative, average or typicalsample of cancer cells, e.g., from a tumor or from affected blood cells,carry at least one copy of the mutation or allele. A cellproliferation-related disorder, characterized by a mutant IDH, e.g., amutant IDH1 or mutant IDH2, having 2HG neoactivity is exemplary. In anembodiment the mutation or allele is present as a heterozygote at theindicated frequencies.

As used herein, a “SNP” is a DNA sequence variation occurring when asingle nucleotide (A, T, C, or G) in the genome (or other sharedsequence) differs between members of a species (or between pairedchromosomes in an individual).

As used herein, a subject can be a human or non-human subject. Non-humansubjects include non-human primates, rodents, e.g., mice or rats, orother non-human animals.

The details of one or more embodiments of the invention are set forth inthe description below. Other features, objects, and advantages of theinvention will be apparent from the description and the drawings, andfrom the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts DNA sequence verification of pET41a-IDH1 and alignmentagainst published IDH1 CDS. The sequence of IDH1 (CDS) corresponds toSEQ ID NO:5. The sequence of pET41a-IDH1 corresponds to SEQ ID NO:6, andthe “consensus” sequence corresponds to SEQ ID NO:7.

FIG. 2 depicts DNA sequence verification of R132S and R132H mutantsaccording to the SEQ ID NO:8. The amino acid sequence of IDH1 (SEQ IDNO:8) is provided in FIG. 21.

FIG. 3 depicts separation of wild type IDH1 protein on Ni-Sepharosecolumn

FIG. 4 depicts protein analysis of wild type IDH1 on SDS gel pre andpost Ni column fractionation. T: total protein; I: insoluble fractions;S: soluble fraction; L: sample for loading on Ni-column. The numbers inthe figure indicates the fraction numbers. Fractions #17˜#27 werecollected for further purification.

FIG. 5A depicts separation of wild type IDH1 protein through SEC columnS-200.

FIG. 5B depicts protein analysis of wild type IDH1 on SDS gel pre andpost S-200 column fractionation. M: molecular weight marker; Ni: nickelcolumn fraction prior to S-200; S200: fraction from SEC column.

FIG. 6 depicts separation of mutant R132S protein on Ni-Sepharosecolumn.

FIG. 7 depicts protein analysis of mutant R132S on SDS gel pre and postNi column fractionation. M: protein marker (KDa): 116, 66.2, 45, 35, 25,18.4, 14.4; T: total cell protein; So: soluble fraction; In: insolublefraction; Ft: flow through. #3-#7 indicate the corresponding elutedfraction numbers.

FIG. 8A depicts separation of mutant R132S protein through SEC columnS-200.

FIG. 8B depicts protein analysis of mutant R132S on SDS gel post S-200column fractionation. M: molecular weight marker; R132S: fraction fromSEC column.

FIG. 9 depicts separation of mutant R132H protein on Ni-Sepharose column

FIG. 10 depicts protein analysis of mutant R132H on SDS gel pre and postNi column fractionation. M: protein marker (KDa): 116, 66.2, 45, 35, 25,18.4, 14.4; T: total cell protein; So: soluble fraction; In: insolublefraction; Ft: flow through; #5-#10 indicate the corresponding elutedfraction numbers; Ni: sample from Ni-Sepharose column, pool #5-#10together.

FIG. 11A depicts separation of mutant R132H protein through SEC columnS-200.

FIG. 11B depicts protein analysis of mutant R132H on SDS gel post S-200column fractionation. M: molecular weight marker; R132H: fraction fromSEC column.

FIG. 12A depicts Michaelis-Menten plot of IDH1 wild-type in theoxidative decarboxylation of ioscitrate to α-ketoglutarate.

FIG. 12B depicts Michaelis-Menten plot of R132H mutant enzyme in theoxidative decarboxylation of ioscitrate to α-ketoglutarate.

FIG. 12C depicts Michaelis-Menten plot of R132S mutant enzyme in theoxidative decarboxylation of ioscitrate to α-ketoglutarate.

FIG. 13A depicts α-KG inhibition of IDH1 wild-type.

FIG. 13B depicts α-KG inhibition of R132H mutant enzyme.

FIG. 13C depicts α-KG inhibition of R132S mutant enzyme.

FIG. 14 depicts IDH1 wt, R132H, and R132S in the conversionα-ketoglutarate to 2-hydroxyglutarate.

FIG. 15A depicts Substrate-Concentration velocity plot for R132H mutantenzyme.

FIG. 15B depicts Substrate-Concentration velocity plot for R132S mutantenzyme.

FIG. 16 depicts IDH1 wt, R132H, and R132S in the conversionα-ketoglutarate to 2-hydroxyglutarate with NADH.

FIG. 17A depicts oxalomalate inhibition to IDH1 wt.

FIG. 17B depicts oxalomalate inhibition to R132H.

FIG. 17C depicts oxalomalate inhibition to R132S.

FIG. 18A depicts LC-MS/MS analysis of the control reaction.

FIG. 18B depicts LC-MS/MS analysis of the reaction containing enzyme.

FIG. 18C depicts LC-MS/MS analysis of the spiked control reaction.

FIG. 19 depicts LC-MS/MS analysis of alpha-hydroxyglutarate.

FIG. 20 depicts LC-MS/MS analysis showing that R132H consumes α-KG toproduce 2-hydroxyglutaric acid.

FIG. 21 depicts the amino acid sequence of IDH1 (SEQ ID NO:13) asdescribed in GenBank Accession No. NP_005887.2 (GI No. 28178825) (recorddated May 10, 2009).

FIG. 21A is the cDNA sequence of IDH1 as presented at GenBank AccessionNo. NM_005896.2 (Record dated May 10, 2009; GI No. 28178824) (SEQ IDNO:8).

FIG. 21B depicts the mRNA sequence of IDH1 as described in GenBankAccession No. NM_005896.2 (Record dated May 10, 2009; GI No. 28178824)(SEQ ID NO:9).

FIG. 22 is the amino acid sequence of IDH2 as presented at GenBankAccession No. NM_002168.2 (Record dated Aug. 16, 2009; GI28178831) (SEQID NO:10).

FIG. 22A is the cDNA sequence of IDH2 as presented at GenBank AccessionNo. NM_002168 (Record dated Aug. 16, 2009; GI28178831) (SEQ ID NO:11).

FIG. 22B is the mRNA sequence of IDH2 as presented at GenBank AccessionNo. NM_002168.2 (Record dated Aug. 16, 2009; GI28178831) (SEQ ID NO:12).

FIG. 23 depicts the progress of forward reactions (isocitrate to α-KG)for the mutant enzyme R132H and R132S.

FIG. 24A depicts LC-MS/MS analysis of derivitized 2-HG racemic mixture.

FIG. 24B depicts LC-MS/MS analysis of derivitized R-2HG standard.

FIG. 24C depicts LC-MS/MS analysis of a coinjection of derivitized 2-HGracemate and R-2-HG standard.

FIG. 24D depicts LC-MS/MS analysis of the deriviatized neoactivityreaction product.

FIG. 24E depicts LC-MS/MS analysis of a coinjection of the neoactivityenzyme reaction product and the R-2-HG standard.

FIG. 24F depicts LC-MS/MS analysis of a coinjection of the neoactivityenzyme reaction product and the 2-HG racemic mixture.

FIG. 25 depicts the inhibitory effect of 2-HG derived from the reductionof α-KG by ICDH1 R132H on the wild-type ICDH1 catalytic oxidativedecarboxylation of isocitrate to α-KG.

FIG. 26A depicts levels of 2-HG in CRL-2610 cell lines expressingwildtype or IDH-1 R132H mutant protein.

FIG. 26B depicts levels of 2-HG in HTB-14 cell lines expressing wildtypeor IDH-1 R132H mutant protein.

FIG. 27 depicts human IDH1 genomic DNA: intron/2^(nd) exon sequence.

FIG. 28 depicts concentrations of 2HG in human malignant gliomascontaining R132 mutations in IDH1. Human glioma samples obtained bysurgical resection were snap frozen, genotyped to stratify as wild-type(WT) (N=10) or carrying an R132 mutant allele (Mutant) (n=12) andmetabolites extracted for LC-MS analysis. Among the 12 mutant tumors, 10carried a R132H mutation, one an R132S mutation, and one an R132Gmutation. Each symbol represents the amount of the listed metabolitefound in each tumor sample. Red lines indicate the group sample means.The difference in 2HG observed between WT and R132 mutant IDH1 mutanttumors was statistically significant by Student's t-test (p<0.0001).There were no statistically significant differences in αKG, malate,fumarate, succinate, or isocitrate levels between the WT and R132 mutantIDH1 tumors.

FIG. 29A depicts the structural analysis of R132H mutant IDH1. On leftis shown an overlay structure of R132H mutant IDH1 and WT IDH1 in the‘closed’ conformation. On the right is shown an overlay structure of WTIDH1 in the ‘open’ conformation with mutant IDH1 for comparison.

FIG. 29B depicts the close-up structural comparison of the R132H IDH1(left) and wild-type (WT) IDH1 (right) active-site containing both αKGand NADPH. In addition to changes at residue 132, the position of thecatalytic residues Tyr 139 and Lys 212 are different and αKG is orienteddifferently relative to NADPH for catalytic hydride transfer in the WTversus R132H mutant enzymes.

FIG. 30A depicts the enzymatic properties of IDH1 R132H mutants whenrecombinant human wild-type (WT) and R132H mutant (R132H) IDH1 enzymeswere assessed for oxidative decarboxylation of isocitrate to αKG withNADP as cofactor. Different concentrations of enzyme were used togenerate the curves.

FIG. 30B depicts the enzymatic properties of IDH R132 mutants when WTand R132H mutant IDH1 enzymes were assessed for reduction of αKG withNADPH as cofactor. Different concentrations of enzyme were used togenerate the curves.

FIG. 30C depicts kinetic parameters of oxidative and reductive reactionsas measured for WT and R132H IDH1 enzymes are shown. K_(m) and k_(cat)values for the reductive activity of the WT enzyme were unable to bedetermined as no measurable enzyme activity was detectable at anysubstrate concentration.

FIG. 31A depicts the LC-MS/MS analysis identifying 2HG as the reductivereaction product of recombinant human R132H mutant IDH1.

FIG. 31B depicts the diacetyl-L-tartaric anhydride derivatization andLC-MS/MS analysis of the chirality of 2HG produced by R132H mutant IDH1.Normalized LC-MS/MS signal for the reductive reaction (rxn) productalone, an R(−)-2HG standard alone, and the two together (Rxn+R(−)-2HG)are shown as is the signal for a racemic mixture of R(−) and S(+) forms(2HG Racemate) alone or with the reaction products (Rxn+Racemate).

FIG. 32A depicts SDS-PAGE and Western blot analyses of C-terminalaffinity-purification tagged IDH1 R132S protein used forcrystallization.

FIG. 32B depicts the chromatogram of FPLC analysis of the IDH1 R132Sprotein sample.

FIG. 33 depicts crystals obtained from a protein solution contained 5 mMNADP, 5 mM isocitrate, 10 mM Ca2+. Precipitant solution contained 100 mMMES (pH 6.0) and 20% PEG 6000 using a hanging drop method ofcrystallization.

FIG. 34 depicts crystal obtained from a protein solution contained 5 mMNADP, 5 mM α-ketoglutarate, 10 mM Ca2+. Precipitant contained 100 mM MES(pH 6.5) and 12% PEG 20000.

FIG. 35 is a bar graph depicting elevated NADPH reductive catalysisactivity in IDH2-R172K mutant enzyme as compared to wildtype IDH2.

FIGS. 36A-C are graphs depicting the following: (A) Extracts from IDH1/2wt (n=10), and IDH1/2 mutant (n=16) patient leukemia cells obtained atpresentation and relapse, and IDH1 R132 mutant leukemia cells grown inculture for 14 days (n=14) analyzed by LC-MS to measure levels of 2-HG;and (B) 2-HG measured in serum of patients with IDH1 wt or IDH1 R132mutant leukemia. In (A) and (B), each point represents an individualpatient sample. Diamonds represent wildtype, circles represent IDH1mutants, and triangles represent IDH2 mutants. Horizontal bars indicatethe mean. (*) indicates a statistically significant difference relativeto wild-type patient cells (p<0.05). (C) depicts In vitro growth curvesof IDH1 R132 mutant and IDH1 wild-type AML cells.

FIG. 37 is a graph depicting the results of extracts from leukemia cellsof AML patients carrying an IDH1/2 mutant (n=16) or wild-type (n=10)allele obtained at initial presentation and relapse assayed by LC-MS forlevels of α-KG, succinate, malate, and fumarate. Each point representsan individual patient sample. Open circles represent wild-types, closedcircles represent IDH1 mutants, and triangles represent IDH2 mutants.Horizontal bars represent the mean. There were no statisticallysignificant differences between the wild-type and IDH1/2 mutant AMLsamples.

FIG. 38 depicts graphical representations of LC-MS analysis of in vitroreactions using recombinant IDH1 R132C and IDH2 R172K confirming that2-HG and not isocitrate is the end product of the mutant enzymereactions.

FIGS. 39A and B depict (A) the wild-type IDH1 enzyme catalysis of theoxidative decarboxylation of isocitrate to alpha-ketoglutarate with theconcomitant reduction of NADP to NADPH; and (B) the IDH1 R132C mutantreduction of alpha-ketoglutarate to 2-hydroxyglutarate while oxidizingNADPH to NADP. These are referred to as the “forward” and “partialreverse” reactions, respectively.

DETAILED DESCRIPTION

The inventors have discovered that certain mutated forms of an enzyme(e.g., IDH1 or IDH2) have a gain of function, referred to herein as aneoactivity, which can be targeted in the treatment of a cellproliferation-related disorder, e.g., a proliferative disorder such ascancer. For example, in the case of a metabolic pathway enzyme, a gainof function or neoactivity can serve as a target for treatment ofcancer. Described herein are methods and compositions for the treatmentof a cell proliferation-related disorder, e.g., a proliferative disordersuch as cancer. The methods include, e.g., treating a subject having aglioma or brain tumor characterized by a preselected IDH1 allele, e.g.,an allele having A at position 394, such as a C394A, a C394G, a C394T, aG395C, a G395T or a G395A mutation, (e.g., a C394A mutant) or an A atposition 395 (e.g., a G395A mutant) according to the sequence of SEQ IDNO:5, that encodes an IDH1 having His, Ser, Cys, Gly, Val, Pro or Leu atposition 132 (e.g., His); or a preselected IDH2 allele that encodes anIDH2 having Lys, Gly, Met, Trp, Thr, or Ser at position 172 and having aneoactivity disclosed herein, by administering to the subject atherapeutically effective amount of an inhibitor of IDH1 or IDH2 (e.g.,IDH1), e.g., a small molecule or nucleic acid. The nucleic acid basedinhibitor is, for example, a dsRNA, e.g., a dsRNA that comprises theprimary sequences of the sense strand and antisense strands of Tables7-14. The dsRNA is composed of two separate strands, or a single strandfolded to form a hairpin structure (e.g., a short hairpin RNA (shRNA)).In some embodiments, the nucleic acid based inhibitor is an antisensenucleic acid, such as an antisense having a sequence that overlaps, orincludes, an antisense sequence provided in Tables 7-14.

Neoactivity of an Enzyme

Neoactivity, as used herein, means an activity that arises as a resultof a mutation, e.g., a point mutation, e.g., a substitution, e.g., inthe active site of an enzyme. In an embodiment the neoactivity issubstantially absent from wild type or non-mutant enzyme. This issometimes referred to herein as a first degree neoactivity. An exampleof a first degree neoactivity is a “gain of function” wherein the mutantenzyme gains a new catalytic activity. In an embodiment the neoactivityis present in wild type or non-mutant enzyme but at a level which isless than 10, 5, 1, 0.1, 0.01 or 0.001% of what is seen in the mutantenzyme. This is sometimes referred to herein as a second degreeneoactivity. An example of a second degree neoactivity is a “gain offunction” wherein the mutant enzyme has an increase, for example, a 5fold increase in the rate of a catalytic activity possessed by theenzyme when lacking the mutation.

In some embodiments, a non-mutant form the enzyme, e.g., a wild typeform, converts substance A (e.g., isocitrate) to substance B (e.g.,α-ketoglutarate), and the neoactivity converts substance B (e.g.,α-ketoglutarate) to substance C, sometimes referred to as theneoactivity product (e.g., 2-hydroxyglutarate, e.g.,R-2-hydroxyglutarate). In some embodiments, the enzyme is in a metabolicpathway, e.g., a metabolic pathway leading to fatty acid biosynthesis,glycolysis, glutaminolysis, the pentose phosphate shunt, the nucleotidebiosynthetic pathway, or the fatty acid biosynthetic pathway, e.g., IDH1or IDH2.

In some embodiments, a non-mutant form the enzyme, e.g., a wild typeform, converts substance A to substance B, and the neoactivity convertssubstance B to substance A. In some embodiments, the enzyme is in ametabolic pathway, e.g., a metabolic pathway leading to fatty acidbiosynthesis, glycolysis, glutaminolysis, the pentose phosphate shunt,the nucleotide biosynthetic pathway, or the fatty acid biosyntheticpathway.

Isocitrate Dehydrogenases

Isocitrate dehydrogenases (IDHs) catalyze the oxidative decarboxylationof isocitrate to 2-oxoglutarate (i.e., α-ketoglutarate). These enzymesbelong to two distinct subclasses, one of which utilizes NAD(+) as theelectron acceptor and the other NADP(+). Five isocitrate dehydrogenaseshave been reported: three NAD(+)-dependent isocitrate dehydrogenases,which localize to the mitochondrial matrix, and two NADP(+)-dependentisocitrate dehydrogenases, one of which is mitochondrial and the otherpredominantly cytosolic. Each NADP(+)-dependent isozyme is a homodimer.

IDH1 (isocitrate dehydrogenase 1 (NADP+), cytosolic) is also known asIDH; IDP; IDCD; IDPC or PICD. The protein encoded by this gene is theNADP(+)-dependent isocitrate dehydrogenase found in the cytoplasm andperoxisomes. It contains the PTS-1 peroxisomal targeting signalsequence. The presence of this enzyme in peroxisomes suggests roles inthe regeneration of NADPH for intraperoxisomal reductions, such as theconversion of 2, 4-dienoyl-CoAs to 3-enoyl-CoAs, as well as inperoxisomal reactions that consume 2-oxoglutarate, namely thealpha-hydroxylation of phytanic acid. The cytoplasmic enzyme serves asignificant role in cytoplasmic NADPH production.

The human IDH1 gene encodes a protein of 414 amino acids. The nucleotideand amino acid sequences for human IDH1 can be found as GenBank entriesNM_005896.2 and NP_005887.2 respectively. The nucleotide and amino acidsequences for IDH1 are also described in, e.g., Nekrutenko et al., Mol.Biol. Evol. 15:1674-1684 (1998); Geisbrecht et al., J. Biol. Chem.274:30527-30533 (1999); Wiemann et al., Genome Res. 11:422-435 (2001);The MGC Project Team, Genome Res. 14:2121-2127 (2004); Lubec et al.,Submitted (December-2008) to UniProtKB; Kullmann et al., Submitted(June-1996) to the EMBL/GenBank/DDBJ databases; and Sjoeblom et al.,Science 314:268-274 (2006).

IDH2 (isocitrate dehydrogenase 2 (NADP+), mitochondrial) is also knownas IDH; IDP; IDHM; IDPM; ICD-M; or mNADP-IDH. The protein encoded bythis gene is the NADP(+)-dependent isocitrate dehydrogenase found in themitochondria. It plays a role in intermediary metabolism and energyproduction. This protein may tightly associate or interact with thepyruvate dehydrogenase complex. Human IDH2 gene encodes a protein of 452amino acids. The nucleotide and amino acid sequences for IDH2 can befound as GenBank entries NM 002168.2 and NP 002159.2 respectively. Thenucleotide and amino acid sequence for human IDH2 are also described in,e.g., Huh et al., Submitted (November-1992) to the EMBL/GenBank/DDBJdatabases; and The MGC Project Team, Genome Res. 14:2121-2127 (2004).

Non-mutant, e.g., wild type, IDH1 catalyzes the oxidativedecarboxylation of ioscitrate to α-ketoglutarate thereby reducing NAD⁺(NADP⁺) to NADP (NADPH), e.g., in the forward reaction:

Isocitrate+NAD⁺ (NADP⁺)→α-KG+CO₂+NADH (NADPH)+H⁺

In some embodiments, the neoactivity of a mutant IDH1 can have theability to convert α-ketoglutarate to 2-hydroxyglutarate, e.g.,R-2-hydroxyglutarate:

α-KG+NADH (NADPH)+H⁺→2-hydroxyglutarate, e.g., R-2-hydroxyglutarate+NAD⁺(NADP⁺).

In some embodiments, the neoactivity can be the reduction of pyruvate ormalate to the corresponding α-hydroxyl compounds.

In some embodiments, the neoactivity of a mutant IDH1 can arise from amutant IDH1 having a His, Ser, Cys, Gly, Val, Pro or Leu, or any othermutations described in Yan et al., at residue 132 (e.g., His, Ser, Cys,Gly, Val or Leu; or His, Ser, Cys or Lys). In some embodiments, theneoactivity of a mutant IDH2 can arise from a mutant IDH2 having a Lys,Gly, Met, Trp, Thr, or Ser (e.g., Lys, Gly, Met, Trp, or Ser; or Gly,Met or Lys), or any other mutations described in Yan H et al., atresidue 172. Exemplary mutations include the following: R132H, R132C,R132S, R132G, R132L, and R132V.

In some embodiments, the mutant IDH1 and/or IDH2 (e.g., a mutant IDH1and/or IDH2 having a neoactivity described herein) could lead to anincreased level of 2-hydroxyglutarate, e.g., R-2-hydroxyglutarate in asubject. The accumulation of 2-hydroxyglutarate, e.g.,R-2-hydroxyglutarate in a subject, e.g., in the brain of a subject, canbe harmful. For example, in some embodiments, elevated levels of2-hydroxyglutarate, e.g., R-2-hydroxyglutarate can lead to and/or bepredictive of cancer in a subject such as a cancer of the centralnervous system, e.g., brain tumor, e.g., glioma, e.g., glioblastomamultiforme (GBM). Accordingly, in some embodiments, a method describedherein includes administering to a subject an inhibitor of theneoactivity.

Detection of 2-hydroxyglutarate

2-hydroxyglutarate can be detected, e.g., by LC/MS. To detect secreted2-hydroxyglutarate in culture media, 500 μL aliquots of conditionedmedia can be collected, mixed 80:20 with methanol, and centrifuged at3,000 rpm for 20 minutes at 4 degrees Celsius. The resulting supernatantcan be collected and stored at −80 degrees Celsius prior to LC-MS/MS toassess 2-hydroxyglutarate levels. To measure whole-cell associatedmetabolites, media can be aspirated and cells can be harvested, e.g., ata non-confluent density. A variety of different liquid chromatography(LC) separation methods can be used. Each method can be coupled bynegative electrospray ionization (ESI, −3.0 kV) to triple-quadrupolemass spectrometers operating in multiple reaction monitoring (MRM) mode,with MS parameters optimized on infused metabolite standard solutions.Metabolites can be separated by reversed phase chromatography using 10mM tributyl-amine as an ion pairing agent in the aqueous mobile phase,according to a variant of a previously reported method (Luo et al. JChromatogr A 1147, 153-64, 2007). One method allows resolution of TCAmetabolites: t=0, 50% B; t=5, 95% B; t=7, 95% B; t=8, 0% B, where Brefers to an organic mobile phase of 100% methanol. Another method isspecific for 2-hydroxyglutarate, running a fast linear gradient from50%-95% B (buffers as defined above) over 5 minutes. A Synergi Hydro-RP,100 mm×2 mm, 2.1 μm particle size (Phenomonex) can be used as thecolumn, as described above. Metabolites can be quantified by comparisonof peak areas with pure metabolite standards at known concentration.Metabolite flux studies from ¹³C-glutamine can be performed asdescribed, e.g., in Munger et al. Nat Biotechnol 26, 1179-86, 2008.

In an embodiment 2HG, e.g., R-2HG, is evaluated and the analyte on whichthe determination is based is 2HG, e.g., R-2HG. In an embodiment theanalyte on which the determination is based is a derivative of 2HG,e.g., R-2HG, formed in process of performing the analytic method. By wayof example such a derivative can be a derivative formed in MS analysis.Derivatives can include a salt adduct, e.g., a Na adduct, a hydrationvariant, or a hydration variant which is also a salt adduct, e.g., a Naadduct, e.g., as formed in MS analysis. Exemplary 2HG derivativesinclude dehydrated derivatives such as the compounds provided below or asalt adduct thereof:

Methods of Evaluating Samples and/or Subjects

This section provides methods of obtaining and analyzing samples and ofanalyzing subjects.

Embodiments of the method comprise evaluation of one or more parametersrelated to IDH, e.g., IDH1 or IDH2, an alpha hydroxy neoactivity, e.g.,2HG neoactivity, e.g., to evaluate the IDH1 or IDH2 2HG neoactivitygenotype or phenotype. The evaluation can be performed, e.g., to select,diagnose or prognose the subject, to select a therapeutic agent, e.g.,an inhibitor, or to evaluate response to the treatment or progression ofdisease. In an embodiment the evaluation, which can be performed beforeand/or after treatment has begun, is based, at least in part, onanalysis of a tumor sample, cancer cell sample, or precancerous cellsample, from the subject. E.g., a sample from the patient can beanalyzed for the presence or level of an alpha hydroxy neoactivityproduct, e.g., 2HG, e.g., R-2HG, by evaluating a parameter correlated tothe presence or level of an alpha hydroxy neoactivity product, e.g.,2HG, e.g., R-2HG. An alpha hydroxy neoactivity product, e.g., 2HG, e.g.,R-2HG, in the sample can be determined by a chromatographic method,e.g., by LC-MS analysis. It can also be determined by contact with aspecific binding agent, e.g., an antibody, which binds the alpha hydroxyneoactivity product, e.g., 2HG, e.g., R-2HG, and allows detection. In anembodiment the sample is analyzed for the level of neoactivity, e.g., analpha hydroxy neoactivity, e.g., 2HG neoactivity. In an embodiment thesample is analysed for the presence of a mutant IDH, e.g., IDH1 or IDH2,protein having an alpha hydroxy neoactivity, e.g., 2HG neoactivity (or acorresponding RNA). E.g., a mutant protein specific reagent, e.g., anantibody that specifically binds an IDH mutant protein, e.g., anantibody that specifically binds an IDH1-R132H mutant protein or anIDH2-R172 mutant protein (e.g., an IDH1-R132H mutant protein), can beused to detect neoactive mutant enzyme In an embodiment a nucleic acidfrom the sample is sequenced to determine if a selected allele ormutation of IDH1 or IDH2 disclosed herein is present. In an embodimentthe analysis is other than directly determining the presence of a mutantIDH, e.g., IDH1 or IDH2, protein (or corresponding RNA) or sequencing ofan IDH, e.g., IDH1 or IDH2 gene. In an embodiment the analysis is otherthan directly determining, e.g., it is other than sequencing genomic DNAor cDNA, the presence of a mutation at residue 132 of IDH1 and/or amutation at residue 172 of IDH2. E.g., the analysis can be the detectionof an alpha hydroxy neoactivity product, e.g., 2HG, e.g., R-2HG, or themeasurement of the mutation's an alpha hydroxy neoactivity, e.g., 2HGneoactivity. In an embodiment the sample is removed from the patient andanalyzed. In an embodiment the evaluation can include one or more ofperforming the analysis of the sample, requesting analysis of thesample, requesting results from analysis of the sample, or receiving theresults from analysis of the sample. (Generally herein, analysis caninclude one or both of performing the underlying method or receivingdata from another who has performed the underlying method.)

In an embodiment the evaluation, which can be performed before and/orafter treatment has begun, is based, at least in part, on analysis of atissue (e.g., a tissue other than a tumor sample), or bodily fluid, orbodily product. Exemplary tissues include lymph node, skin, hairfollicles and nails. Exemplary bodily fluids include blood, plasma,urine, lymph, tears, sweat, saliva, semen, and cerebrospinal fluid.Exemplary bodily products include exhaled breath. E.g., the tissue,fluid or product can be analyzed for the presence or level of an alphahydroxy neoactivity product, e.g., 2HG, e.g., R-2HG, by evaluating aparameter correlated to the presence or level of an alpha hydroxyneoactivity product, e.g., 2HG, e.g., R-2HG. An alpha hydroxyneoactivity product, e.g., 2HG, e.g., R-2HG, in the sample can bedetermined by a chromatographic method, e.g., by LC-MS analysis. It canalso be determined by contact with a specific binding agent, e.g., anantibody, which binds the alpha hydroxy neoactivity product, e.g., 2HG,e.g., R-2HG, and allows detection. In embodiments where sufficientlevels are present, the tissue, fluid or product can be analyzed for thelevel of neoactivity, e.g., an alpha hydroxy neoactivity, e.g., the 2HGneoactivity. In an embodiment the sample is analysed for the presence ofa mutant IDH, e.g., IDH1 or IDH2, protein having an alpha hydroxyneoactivity, e.g., 2HG neoactivity (or a corresponding RNA). E.g., amutant protein specific reagent, e.g., an antibody that specificallybinds an IDH mutant protein, e.g., an antibody that specifically bindsan IDH1-R132H mutant protein or an IDH2-R172 mutant protein (e.g., anIDH1-R132H mutant protein), can be used to detect neoactive mutantenzyme. In an embodiment a nucleic acid from the sample is sequenced todetermine if a selected allele or mutation of IDH1 or IDH2 disclosedherein is present. In an embodiment the analysis is other than directlydetermining the presence of a mutant IDH, e.g., IDH1 or IDH2, protein(or corresponding RNA) or sequencing of an IDH, e.g., IDH1 or IDH2 gene.E.g., the analysis can be the detection of an alpha hydroxy neoactivityproduct, e.g., 2HG, e.g., R-2HG, or the measurement of 2HG neoactivity.In an embodiment the tissue, fluid or product is removed from thepatient and analyzed. In an embodiment the evaluation can include one ormore of performing the analysis of the tissue, fluid or product,requesting analysis of the tissue, fluid or product, requesting resultsfrom analysis of the tissue, fluid or product, or receiving the resultsfrom analysis of the tissue, fluid or product.

In an embodiment the evaluation, which can be performed before and/orafter treatment has begun, is based, at least in part, on alpha hydroxyneoactivity product, e.g., 2HG, e.g., R-2HG, imaging of the subject. Inembodiments magnetic resonance methods are is used to evaluate thepresence, distribution, or level of an alpha hydroxy neoactivityproduct, e.g., 2HG, e.g., R-2HG, in the subject. In an embodiment thesubject is subjected to imaging and/or spectroscopic analysis, e.g.,magnetic resonance-based analysis, e.g., MRI and/or MRS e.g., analysis,and optionally an image corresponding to the presence, distribution, orlevel of an alpha hydroxy neoactivity product, e.g., 2HG, e.g., R-2HG,or of the tumor, is formed. Optionally the image or a value related tothe image is stored in a tangible medium and/or transmitted to a secondsite. In an embodiment the evaluation can include one or more ofperforming imaging analysis, requesting imaging analysis, requestingresults from imaging analysis, or receiving the results from imaginganalysis.

Methods of Treating a Proliferative Disorder

Described herein are methods of treating a cell proliferation-relateddisorder, e.g., a cancer, e.g., a glioma, e.g., by inhibiting aneoactivity of a mutant enzyme, e.g., an enzyme in a metabolic pathway,e.g., a metabolic pathway leading to fatty acid biosynthesis,glycolysis, glutaminolysis, the pentose phosphate shunt, the nucleotidebiosynthetic pathway, or the fatty acid biosynthetic pathway, e.g., IDH1or IDH2. The cancer can be characterized by the presence of aneoactivity, such as a gain of function in one or more mutant enzymes(e.g., an enzyme in the metabolic pathway, e.g., a metabolic pathwayleading to fatty acid biosynthesis, glycolysis, glutaminolysis, thepentose phosphate shunt, the nucleotide biosynthetic pathway, or thefatty acid biosynthetic pathway e.g., IDH1 or IDH2). In someembodiments, the gain of function is the conversion of α-ketoglutarateto 2-hydroxyglutarate, e.g., R-2-hydroxyglutarate.

Compounds for the Treatment of Cancer

A candidate compound can be evaluated for modulation (e.g., inhibition)of neoactivity, for example, using an assay described herein. Acandidate compound can also be evaluated for modulation (e.g.,inhibition) of wild type or non-mutant activity. For example, theformation of a product or by-product of any activity (e.g., enzymaticactivity) can be assayed, thus evaluating a candidate compound. In someembodiments, the activity (e.g., wild type/non-mutant or neoactivity)can be evaluated by measuring one or more readouts from an enzymaticassay. For example, the change in nature and/or amount of substrateand/or product can be measured, e.g., using methods such as fluorescentor radiolabeled substrates. Exemplary substrates and/or products includeα-ketoglutarate, CO₂, NADP, NADPH, NAD, NADH, and 2-hydroxyglutarate,e.g., R-2-hydroxyglutarate. In some embodiments, the rate of reaction ofthe enzyme can also be evaluated as can the nature and/or amount of aproduct of the enzymatic reaction. In addition to the measurement ofpotential enzymatic activities, activity (e.g., wild type/non-mutant orneoactivity) can be detected by the quenching of protein fluorescenceupon binding of a potential substrate, cofactor, or enzymatic activitymodulator to the enzyme.

In one embodiment, assay progress can be monitored by changes in theOD340 or fluorescence of the NAD or NADP cofactor. In anotherembodiment, the reaction progress can be coupled to a secondary enzymeassay system in continuous mode or endpoint mode for increasing thedynamic range of the assay. For example, an endpoint assay can beperformed by adding to the reaction an excess of diaphorase andrezasarin. Diaphorase consumes the remaining NADPH or NADH whileproducing resorufin from rezasarin. Resorufin is a highly fluorescentproduct which can be measured by fluorescence at Ex544 Em590. This notonly terminates the reaction but also generates an easily detectablesignal with greater quantum yield than the fluorescence of the cofactor.

A continuous assay can be implemented through coupling a product of theprimary reaction to a secondary enzyme reaction that yields detectableresults of greater dynamic range or more convenient detection mode. Forexample, inclusion in the reaction mix of aldehyde dehydrogenase (ALDH),which is an NADP+ dependent enzyme, and 6-methoxy-2-napthaldehye, achromogenic substrate for ALDH, will result in the production of thefluorescent product 6-methoxy-2-napthoate (Ex310 Em 360) at a ratedependent on the production of NADP+ by isocitrate dehydrogenase. Theinclusion of a coupling enzyme such as aldehyde dehydrogenase has theadditional benefit of allowing screening of neoactivity irrespective ofwhether NADP+ or NAD+ is produced, since this enzyme is capable ofutilizing both. Additionally, since the NADPH or NADH cofactor requiredfor the “reverse” assay is regenerated, a coupled enzyme system whichcycles the cofactor back to the IDH enzyme has the further advantage ofpermitting continuous assays to be conducted at cofactor concentrationsmuch below Km for the purpose of enhancing the detection of competitiveinhibitors of cofactor binding.

In yet a third embodiment of an activity (e.g., wild type/non-mutant orneoactivity) screen, one or a number of IDH substrates, cofactors, orproducts can be isotopically labeled with radioactive or “heavy”elements at defined atoms for the purpose of following specificsubstrates or atoms of substrates through the chemical reaction. Forexample, the alpha carbon of α-KG, isocitrate, or 2-hydroxyglutarate,e.g., R-2-hydroxyglutarate may be ¹⁴C or ¹³C. Amount, rate, identity andstructure of products formed can be analyzed by means known to those ofskill in the art, for example mass spectroscopy or radiometric HPLC.

Compounds that inhibit a neoactivity, e.g., a neoactivity describedherein, can include, e.g., small molecule, nucleic acid, protein andantibody.

Exemplary small molecules include, e.g, small molecules that bind toenzymes and decrease their activity, e.g., a neoactivity describedherein. The binding of an inhibitor can stop a substrate from enteringthe enzyme's active site and/or hinder the enzyme from catalyzing itsreaction. Inhibitor binding is either reversible or irreversible.Irreversible inhibitors usually react with the enzyme and change itchemically. These inhibitors can modify key amino acid residues neededfor enzymatic activity. In contrast, reversible inhibitors bindnon-covalently and different types of inhibition are produced dependingon whether these inhibitors bind the enzyme, the enzyme-substratecomplex, or both.

In some embodiments, the small molecule is oxalomalate, oxalofumarate,or oxalosuccinate.

In some embodiments, the small molecule is a compound of formula (X), ora compound as listed in Table 24a. The compound of formula (X) isprovided below:

wherein X is C₁-C₆ alkylene (e.g., methylene), C(O), or C(O)C₁-C₆alkylene; wherein X is optionally substituted;

R¹ is halo (e.g., fluoro), C₁-C₆ alkyl, C₁-C₆ haloalkyl, hydroxyl, C₁-C₆alkoxy, cyano, nitro, amino, alkylamino, dialkylamino, amido, —C(O)OH,or C(O)OC₁-C₆alkyl; and

m is 0, 1, 2, or 3.

In some embodiments, the compound is a compound of formula (XI) or apharmaceutically acceptable salt thereof or a compound listed in Table24b

wherein:W, X, Y and Z are each independently selected from CH or N;B and B¹ are independently selected from hydrogen, alkyl or when takentogether with the carbon to which they are attached form a carbonylgroup;

Q is C═O or SO₂;

D and D¹ are independently selected from a bond, oxygen or NR^(c);A is optionally substituted aryl or optionally substituted heteroaryl;R¹ is independently selected from alkyl, acyl, cycloalkyl, aryl,heteroaryl, heterocyclyl, heterocyclylalkyl, cycloalkylalkyl, aralkyl,and heteroaralkyl; each of which may be optionally substituted with 0-3occurrences of R^(d);each R³ is independently selected from halo, haloalkyl, alkyl and—OR^(a);each R^(a) is independently selected from alkyl, and haloalkyl;each R^(b) is independently alkyl;each R^(c) is independently selected from hydrogen, alkyl and alkenyl;each R^(d) is independently selected from halo, haloalkyl, alkyl, nitro,cyano, and —OR^(a), or two R^(d) taken together with the carbon atoms towhich they are attached form an optionally substituted heterocyclyl;n is 0, 1, or 2;h is 0, 1, 2; andg is 0, 1 or 2.

In some embodiments, the small molecule is a selective inhibitor of theneoactivity (e.g., relative to the wild type activity).

Nucleic acids can be used to inhibit a neoactivity, e.g., a neoactivitydescribed herein, e.g., by decreasing the expression of the enzyme.Exemplary nucleic acids include, e.g., siRNA, shRNA, antisense RNA,aptamer and ribozyme. Art-known methods can be used to select inhibitorymolecules, e.g., siRNA molecules, for a particular gene sequence.

Proteins can also be used to inhibit a neoactivity, e.g., a neoactivitydescribed herein, by directly or indirectly binding to the enzyme and/orsubstrate, or competing binding to the enzyme and/or substrate.Exemplary proteins include, e.g., soluble receptors, peptides andantibodies. Exemplary antibodies include, e.g., whole antibody or afragment thereof that retains its ability to bind to the enzyme orsubstrate.

Exemplary candidate compounds, which can be tested for inhibition of aneoactivity described herein (e.g., a neoactivity associated with mutantIDH1), are described in the following references, each of which areincorporated herein by reference: Bioorganic & Medicinal Chemistry(2008), 16(7), 3580-3586; Free Radical Biology & Medicine (2007), 42(1),44-51; KR 2005036293 A; Applied and Environmental Microbiology (2005),71(9), 5465-5475; KR 2002095553 A; U.S. Pat. Appl. US 2004067234 A1; PCTInt. Appl. (2002), WO 2002033063 A1; Journal of Organic Chemistry(1996), 61(14), 4527-4531; Biochimica et Biophysica Acta, Enzymology(1976), 452(2), 302-9; Journal of Biological Chemistry (1975), 250(16),6351-4; Bollettino—Societa Italiana di Biologia Sperimentale (1972),48(23), 1031-5; Journal of Biological Chemistry (1969), 244(20),5709-12.

Isomers

Certain compounds may exist in one or more particular geometric,optical, enantiomeric, diasteriomeric, epimeric, atropic, stereoisomer,tautomeric, conformational, or anomeric forms, including but not limitedto, cis- and trans-forms; E- and Z-forms; c-, t-, and r-forms; endo- andexo-forms; R-, S-, and meso-forms; D- and L-forms; d- and l-forms; (+)and (−) forms; keto-, enol-, and enolate-forms; syn- and anti-forms;synclinal- and anticlinal-forms; α- and β-forms; axial and equatorialforms; boat-, chair-, twist-, envelope-, and halfchair-forms; andcombinations thereof, hereinafter collectively referred to as “isomers”(or “isomeric forms”).

In one embodiment, a compound described herein, e.g., an inhibitor of aneoactivity or 2-HG is an enantiomerically enriched isomer of astereoisomer described herein. For example, the compound has anenantiomeric excess of at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or99%. Enantiomer, when used herein, refers to either of a pair ofchemical compounds whose molecular structures have a mirror-imagerelationship to each other.

In one embodiment, a preparation of a compound disclosed herein isenriched for an isomer of the compound having a selectedstereochemistry, e.g., R or S, corresponding to a selected stereocenter,e.g., the 2-position of 2-hydroxyglutaric acid. 2HG can be purchasedfrom commercial sources or can be prepared using methods known in theart, for example, as described in Org. Syn. Coll vol., 7, P-99, 1990.For example, the compound has a purity corresponding to a compoundhaving a selected stereochemistry of a selected stereocenter of at leastabout 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%.

In one embodiment, a composition described herein includes a preparationof a compound disclosed herein that is enriched for a structure orstructures having a selected stereochemistry, e.g., R or S, at aselected stereocenter, e.g., the 2-position of 2-hydroxyglutaric acid.Exemplary R/S configurations can be those provided in an exampledescribed herein.

An “enriched preparation,” as used herein, is enriched for a selectedstereoconfiguration of one, two, three or more selected stereocenterswithin the subject compound. Exemplary selected stereocenters andexemplary stereoconfigurations thereof can be selected from thoseprovided herein, e.g., in an example described herein. By enriched ismeant at least 60%, e.g., of the molecules of compound in thepreparation have a selected stereochemistry of a selected stereocenter.In an embodiment it is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%,97%, 98%, or 99%. Enriched refers to the level of a subject molecule(s)and does not connote a process limitation unless specified.

Note that, except as discussed below for tautomeric forms, specificallyexcluded from the term “isomers,” as used herein, are structural (orconstitutional) isomers (i.e., isomers which differ in the connectionsbetween atoms rather than merely by the position of atoms in space). Forexample, a reference to a methoxy group, —OCH3, is not to be construedas a reference to its structural isomer, a hydroxymethyl group, —CH2OH.Similarly, a reference to ortho-chlorophenyl is not to be construed as areference to its structural isomer, meta-chlorophenyl. However, areference to a class of structures may well include structurallyisomeric forms falling within that class (e.g., C1-7alkyl includesn-propyl and iso-propyl; butyl includes n-, iso-, sec-, and tert-butyl;methoxyphenyl includes ortho-, meta-, and para-methoxyphenyl).

The above exclusion does not pertain to tautomeric forms, for example,keto-, enol-, and enolate-forms, as in, for example, the followingtautomeric pairs: keto/enol (illustrated below), imine/enamine,amide/imino alcohol, amidine/amidine, nitroso/oxime,thioketone/enethiol, N-nitroso/hydroxyazo, and nitro/aci-nitro.

Note that specifically included in the term “isomer” are compounds withone or more isotopic substitutions. For example, H may be in anyisotopic form, including 1H, 2H (D), and 3H (T); C may be in anyisotopic form, including 12C, 13C, and 14C; O may be in any isotopicform, including 16O and 18O; and the like. Unless otherwise specified, areference to a particular compound includes all such isomeric forms,including (wholly or partially) racemic and other mixtures thereof.Methods for the preparation (e.g., asymmetric synthesis) and separation(e.g., fractional crystallisation and chromatographic means) of suchisomeric forms are either known in the art or are readily obtained byadapting the methods taught herein, or known methods, in a known manner.

Salts

It may be convenient or desirable to prepare, purify, and/or handle acorresponding salt of the active compound, for example, apharmaceutically-acceptable salt. Examples of pharmaceuticallyacceptable salts are discussed in Berge et al., 1977, “PharmaceuticallyAcceptable Salts.” J. Pharm. ScL. Vol. 66, pp. 1-19.

For example, if the compound is anionic, or has a functional group whichmay be anionic (e.g., —COOH may be —COO″), then a salt may be formedwith a suitable cation. Examples of suitable inorganic cations include,but are not limited to, alkali metal ions such as Na+ and K+, alkalineearth cations such as Ca2+ and Mg2+, and other cations such as Al+3.Examples of suitable organic cations include, but are not limited to,ammonium ion (i.e., NH4+) and substituted ammonium ions (e.g., NH3R+,NH2R2+, NHR3+, NR4+). Examples of some suitable substituted ammoniumions are those derived from: ethylamine, diethylamine,dicyclohexylamine, triethylamine, butylamine, ethylenediamine,ethanolamine, diethanolamine, piperazine, benzylamine,phenylbenzylamine, choline, meglumine, and tromethamine, as well asamino acids, such as lysine and arginine. An example of a commonquaternary ammonium ion is N(CH3)4+.

If the compound is cationic, or has a functional group that may becationic (e.g., —NH2 may • be —NH3+), then a salt may be formed with asuitable anion. Examples of suitable inorganic anions include, but arenot limited to, those derived from the following inorganic acids:hydrochloric, hydrobromic, hydroiodic, sulfuric, sulfurous, nitric,nitrous, phosphoric, and phosphorous.

Examples of suitable organic anions include, but are not limited to,those derived from the following organic acids: 2-acetyoxybenzoic,acetic, ascorbic, aspartic, benzoic, camphorsulfonic, cinnamic, citric,edetic, ethanedisulfonic, ethanesulfonic, fumaric, glucheptonic,gluconic, glutamic, glycolic, hydroxymaleic, hydroxynaphthalenecarboxylic, isethionic, lactic, lactobionic, lauric, maleic, malic,methanesulfonic, mucic, oleic, oxalic, palmitic, pamoic, pantothenic,phenylacetic, phenylsulfonic, propionic, pyruvic, salicylic, stearic,succinic, sulfanilic, tartaric, toluenesulfonic, and valeric. Examplesof suitable polymeric organic anions include, but are not limited to,those derived from the following polymeric acids: tannic acid,carboxymethyl cellulose.

Unless otherwise specified, a reference to a particular compound alsoincludes salt forms thereof.

Chemically Protected Forms

It may be convenient or desirable to prepare, purify, and/or handle theactive compound in a chemically protected form. The term “chemicallyprotected form” is used herein in the conventional chemical sense andpertains to a compound in which one or more reactive functional groupsare protected from undesirable chemical reactions under specifiedconditions (e.g., pH, temperature, radiation, solvent, and the like). Inpractice, well known chemical methods are employed to reversibly renderunreactive a functional group, which otherwise would be reactive, underspecified conditions. In a chemically protected form, one or morereactive functional groups are in the form of a protected or protectinggroup (also known as a masked or masking group or a blocked or blockinggroup). By protecting a reactive functional group, reactions involvingother unprotected reactive functional groups can be performed, withoutaffecting the protected group; the protecting group may be removed,usually in a subsequent step, without substantially affecting theremainder of the molecule. See, for example, Protective Groups inOrganic Synthesis (T. Green and P. Wuts; 3rd Edition; John Wiley andSons, 1999). Unless otherwise specified, a reference to a particularcompound also includes chemically protected forms thereof.

A wide variety of such “protecting,” “blocking,” or “masking” methodsare widely used and well known in organic synthesis. For example, acompound which has two nonequivalent reactive functional groups, both ofwhich would be reactive under specified conditions, may be derivatizedto render one of the functional groups “protected,” and thereforeunreactive, under the specified conditions; so protected, the compoundmay be used as a reactant which has effectively only one reactivefunctional group. After the desired reaction (involving the otherfunctional group) is complete, the protected group may be “deprotected”to return it to its original functionality.

For example, a hydroxy group may be protected as an ether (—OR) or anester (—OC(═O)R), for example, as: a t-butyl ether; a benzyl, benzhydryl(diphenylmethyl), or trityl (triphenylmethyl) ether; a trimethylsilyl ort-butyldimethylsilyl ether; or an acetyl ester (—OC(═O)CH3, —OAc).

For example, an aldehyde or ketone group may be protected as an acetal(R—CH(OR)2) or ketal (R2C(OR)2), respectively, in which the carbonylgroup (>C═O) is converted to a diether (>C(OR)2), by reaction with, forexample, a primary alcohol. The aldehyde or ketone group is readilyregenerated by hydrolysis using a large excess of water in the presenceof acid.

For example, an amine group may be protected, for example, as an amide(—NRCO—R) or a urethane (—NRCO—OR), for example, as: a methyl amide(—NHCO—CH3); a benzyloxy amide (—NHCO—OCH2C6H5, —NH-Cbz); as a t-butoxyamide (—NHCO—OC(CH3)3, —NH-Boc); a 2-biphenyl-2-propoxy amide(—NHCO—OC(CH3)2C6H4C6H5, —NH-Bpoc), as a 9-fluorenylmethoxy amide(—NH-Fmoc), as a 6-nitroveratryloxy amide (—NH-Nvoc), as a2-trimethylsilylethyloxy amide (—NH-Teoc), as a 2,2,2-trichloroethyloxyamide (—NH-Troc), as an allyloxy amide (—NH-Alloc), as a2(-phenylsulphonyl)ethyloxy amide (—NH-Psec); or, in suitable cases(e.g., cyclic amines), as a nitroxide radical (>N—O<<).

For example, a carboxylic acid group may be protected as an ester forexample, as: an Ĉalkyl ester (e.g., a methyl ester; a t-butyl ester); aCvrhaloalkyl ester (e.g., a C1-7trihaloalkyl ester); atriC1-7alkylsilyl-Ci.7alkyl ester; or a C5.2oaryl-C1-7alkyl ester (e.g.,a benzyl ester; a nitrobenzyl ester); or as an amide, for example, as amethyl amide.

For example, a thiol group may be protected as a thioether (—SR), forexample, as: a benzyl thioether; an acetamidomethyl ether(—S—CH2NHC(═O)CH3).

Nucleic Acid Based Inhibitors

Nucleic acid-based inhibitors for inhibition IDH, e.g., IDH1, can be,e.g., double stranded RNA (dsRNA) that function, e.g., by an RNAinterference (RNAi mechanism), an antisense RNA, or a microRNA (miRNA).In an embodiment the nucleic-acid based inhibitor binds to the targetmRNA and inhibits the production of protein therefrom, e.g., by cleavageof the target mRNA.

Double Stranded RNA (dsRNA)

A nucleic acid based inhibitor useful for decreasing IDH1 or IDH2 mutantfunction is, e.g., a dsRNA, such as a dsRNA that acts by an RNAimechanism. RNAi refers to the process of sequence-specificpost-transcriptional gene silencing in animals mediated by shortinterfering RNAs (siRNAs). dsRNAs as used herein are understood toinclude siRNAs. Typically, inhibition of IDH, e.g., IDH1, by dsRNAs doesnot trigger the interferon response that results from dsRNA-mediatedactivation of protein kinase PKR and 2′,5′-oligoadenylate synthetaseresulting in non-specific cleavage of mRNA by ribonuclease L.

dsRNAs targeting an IDH, e.g., IDH1, enzyme, e.g., a wildtype or mutantIDH1, can be unmodified or chemically modified. The dsRNA can bechemically synthesized, expressed from a vector or enzymaticallysynthesized. The invention also features various chemically modifiedsynthetic dsRNA molecules capable of modulating IDH1 gene expression oractivity in cells by RNA interference (RNAi). The use of chemicallymodified dsRNA improves various properties of native dsRNA molecules,such as through increased resistance to nuclease degradation in vivoand/or through improved cellular uptake.

The dsRNAs targeting nucleic acid can be composed of two separate RNAs,or of one RNA strand, which is folded to form a hairpin structure.Hairpin dsRNAs are typically referred to as shRNAs.

An shRNA that targets IDH, e.g., a mutant or wildtype IDH1 gene can beexpressed from a vector, e.g., viral vector, such as a lentiviral oradenoviral vector. In certain embodiments, a suitable dsRNA forinhibiting expression of an IDH1 gene will be identified by screening ansiRNA library, such as an adenoviral or lentiviral siRNA library.

In an embodiment, a dsRNA that targets IDH, e.g., IDH1, is about 15 toabout 30 base pairs in length (e.g., about 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, or 29) basepairs in length. In anotherembodiment, the dsRNA includes overhanging ends of about 1 to about 3(e.g., about 1, 2, or 3) nucleotides. By “overhang” is meant that 3′-endof one strand of the dsRNA extends beyond the 5′-end of the otherstrand, or vice versa. The dsRNA can have an overhang on one or bothends of the dsRNA molecule. In some embodiments, the single-strandedoverhang is located at the 3′-terminal end of the antisense strand, or,alternatively, at the 3′-terminal end of the sense strand. In someembodiments, the overhang is a TT or UU dinucleotide overhang, e.g., aTT or UU dinucleotide overhang. For example, in an embodiment, the dsRNAincludes a 21-nucleotide antisense strand, a 19 base pair duplex region,and a 3′-terminal dinucleotide. In yet another embodiment, a dsRNAincludes a duplex nucleic acid where both ends are blunt, oralternatively, where one of the ends is blunt.

In an embodiment, the dsRNA includes a first and a second strand, eachstrand is about 18 to about 28 nucleotides in length, e.g., about 19 toabout 23 nucleotides in length, the first strand of the dsRNA includes anucleotide sequence having sufficient complementarity to the IDH, e.g.,IDH1, RNA for the dsRNA to direct cleavage of the IDH, e.g., IDH1, mRNAvia RNA interference, and the second strand of the dsRNA includes anucleotide sequence that is complementary to the first strand.

In an embodiment, a dsRNA targeting an IDH, e.g., IDH1, gene can targetwildtype and mutant forms of the gene, or can target different allelicisoforms of the same gene. For example, the dsRNA will target a sequencethat is identical in two or more of the different isoforms. In anembodiment, the dsRNA targets an IDH1 having G at position 395 or C atposition 394 (e.g., a wildtype IDH1 RNA) and an IDH1 having A atposition 395 or A at position 394, such as a C394A, a C394G, a C394T, aG395C, a G395T or a G395A mutation, (e.g., an IDH1 RNA carrying a G395Aand/or a C394A mutation) (FIG. 2).

In an embodiment, a dsRNA will preferentially or specifically target amutant IDH RNA, or a particular IDH polymorphism. In some embodiments,the IDH has a mutation at position 394 or 395 such as a C394A, a C394G,a C394T, a G395C, a G395T or a G395A mutation. For example, in anembodiment, the dsRNA targets an IDH1 RNA carrying an A at position 395,e.g., G395A, and in another embodiment, the dsRNA targets an IDH1 RNAcarrying an A at position 394, e.g., C394A mutation.

In an embodiment, a dsRNA targeting an IDH RNA includes one or morechemical modifications. Non-limiting examples of such chemicalmodifications include without limitation phosphorothioateinternucleotide linkages, 2′-deoxyribonucleotides, 2′-O-methylribonucleotides, 2′-deoxy-2′-fluoro ribonucleotides, “universal base”nucleotides, “acyclic” nucleotides, 5-C-methyl nucleotides, and terminalglyceryl and/or inverted deoxy abasic residue incorporation. Suchchemical modifications have been shown to preserve RNAi activity incells while at the same time, dramatically increasing the serumstability of these compounds. Furthermore, one or more phosphorothioatesubstitutions are well-tolerated and have been shown to confersubstantial increases in serum stability for modified dsRNA constructs.

In an embodiment, a dsRNA targeting an IDH, e.g., IDH1, RNA includesmodified nucleotides while maintaining the ability to mediate RNAi. Themodified nucleotides can be used to improve in vitro or in vivocharacteristics such as stability, activity, and/or bioavailability. Forexample, the dsRNA can include modified nucleotides as a percentage ofthe total number of nucleotides present in the molecule. As such, thedsRNA can generally include about 5% to about 100% modified nucleotides(e.g., about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% modified nucleotides).

In some embodiments, the dsRNA targeting IDH, e.g., IDH1, is about 21nucleotides long. In another embodiment, the dsRNA does not contain anyribonucleotides, and in another embodiment, the dsRNA includes one ormore ribonucleotides. In an embodiment, each strand of the dsRNAmolecule independently includes about 15 to about 30 (e.g., about 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30)nucleotides, wherein each strand includes about 15 to about 30 (e.g.,about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30)nucleotides that are complementary to the nucleotides of the otherstrand. In an embodiment, one of the strands of the dsRNA includes anucleotide sequence that is complementary to a nucleotide sequence or aportion thereof of the IDH1 or IDH2 gene, and the second strand of thedsRNA includes a nucleotide sequence substantially similar to thenucleotide sequence of the IDH1 or IDH2 gene or a portion thereof.

In an embodiment, the dsRNA targeting IDH1 or IDH2 includes an antisenseregion having a nucleotide sequence that is complementary to anucleotide sequence of the IDH1 or IDH2 gene or a portion thereof, and asense region having a nucleotide sequence substantially similar to thenucleotide sequence of the IDH1 or IDH2 gene or a portion thereof. In anembodiment, the antisense region and the sense region independentlyinclude about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides, where the antisenseregion includes about 15 to about 30 (e.g., about 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides that arecomplementary to nucleotides of the sense region.

As used herein, the term “dsRNA” is meant to include nucleic acidmolecules that are capable of mediating sequence specific RNAi, such asshort interfering RNA (siRNA), short hairpin RNA (shRNA), shortinterfering oligonucleotide, short interfering nucleic acid, shortinterfering modified oligonucleotide, chemically modified siRNA,post-transcriptional gene silencing RNA (ptgsRNA), and others. Inaddition, as used herein, the term “RNAi” is meant to include sequencespecific RNA interference, such as post transcriptional gene silencing,translational inhibition, or epigenetics.

Nucleic Acid-Based IDH Inhibitors

In an embodiment the inhibitor is a nucleic acid-based inhibitor, suchas a double stranded RNA (dsRNA) or antisense RNA that targets a mutantIDH, e.g., mutant IDH1 or IDH2.

In one embodiment, the nucleic acid based inhibitor, e.g., a dsRNA orantisense molecule, decreases or inhibits expression of an IDH1 havingother than an Arg, e.g., having a His, Ser, Cys, Gly, Val, Pro or Leu,or any residue described in Yan et al., N. Eng. J. Med. 360:765-73, atresidue 132, according to the sequence of SEQ ID NO:8 (see also FIG.21). In one embodiment, the nucleic acid based inhibitor decreases orinhibits expression of an IDH1 enzyme having His at residue 132

In an embodiment the nucleic acid-based inhibitor is a dsRNA thattargets an mRNA that encodes an IDH1 allele described herein, e.g., anIDH1 allele having other than an Arg at residue 132. E.g., the alleleencodes His, Ser, Cys, Gly, Val, Pro or Leu, or any residue described inYan et al., at residue 132, according to the sequence of SEQ ID NO:8(see also FIG. 21).

In an embodiment the allele encodes an IDH1 having His at residue 132.

In an embodiment the allele encodes an IDH1 having Ser at residue 132.

In an embodiment, the nucleic acid-based inhibitor is a dsRNA thattargets IDH1, e.g., an IDH1 having an A or a T (or a nucleotide otherthan C) at nucleotide position 394 or an A (or a nucleotide other thanG) at nucleotide position 395, e.g., a mutant allele carrying a C394Tmutation or a G395A mutation according to the IDH1 sequence of SEQ IDNO:8 (see also FIG. 21A).

In an embodiment, the dsRNA targets an IDH1 having other than C, e.g., aT or an A, at nucleotide position 394 or and other than G, e.g., an A,at 395 (e.g., a mutant) and an IDH1 having a C at nucleotide position394 or a G at nucleotide position 395 (e.g., a wildtype), e.g., bytargeting a region of the IDH1 mRNA that is identical between thewildtype and mutant transcripts. In yet another embodiment, the dsRNAtargets a particular mutant or polymorphism (such as a single nucleotidepolymorphism (SNP)), but not a wildtype allele. In this case, thenucleic acid based inhibitor, e.g., a dsRNA, targets the region of theIDH1 containing the mutation.

In some embodiments, the nucleic acid based inhibitor, e.g., a dsRNApreferentially or specifically inhibits the product of a mutant IDH1 ascompared to the product of a wildtype IDH1. In some embodiments, the IDHhas a mutation at position 394 or 395 such as a C394A, a C394G, a C394T,a G395C, a G395T or a G395A mutation. For example, in one embodiment, adsRNA targets a region of an IDH1 mRNA that carries the mutation (e.g.,a C394A of C394T or a G395A mutation according to SEQ ID NO:5).

In one embodiment, the nucleic acid-based inhibitor is a dsRNA includinga sense strand and an antisense strand having a primary sequencepresented in Tables 7-14. In another embodiment, the nucleic acid basedinhibitor is an antisense oligonucleotide that includes all or a part ofan antisense primary sequence presented in Tables 7-14 or which targetsthe same or substantially the same region as does a dsRNA from Tables7-14.

In one embodiment, the nucleic acid based inhibitor decreases orinhibits expression of an IDH2 having Lys, Gly, Met, Trp, Thr, Ser, orany residue described in Yan et al., at residue 172, according to theamino acid sequence of SEQ ID NO:10 (see also FIG. 22). In oneembodiment, the nucleic acid based inhibitor decreases or inhibitsexpression of an IDH2 enzyme having Lys at residue 172.

In an embodiment the nucleic acid-based inhibitor is a dsRNA thattargets an mRNA that encodes an IDH2 allele described herein, e.g., anIDH2 allele having other than an Arg at residue 172. E.g., the allelecan have Lys, Gly, Met, Trp, Thr, Ser, or any residue described in Yanet al., at residue 172, according to the sequence of SEQ ID NO:10 (seealso FIG. 22).

In an embodiment the allele encodes an IDH2 having Lys at residue 172.

In an embodiment the allele encodes an IDH2 having Met at residue 172.

In an embodiment, the nucleic acid-based inhibitor is a dsRNA thattargets IDH2, e.g., an IDH2 having a G or a T (or a nucleotide otherthan A or C) at nucleotide position 514 or an A or T or C (or anucleotide other than G) at nucleotide position 515, e.g., a mutantallele carrying a A514G mutation or a G515T or a G515A mutationaccording to the IDH2 sequence of SEQ ID NO:10 (FIG. 22A). In oneembodiment, the nucleic acid-based inhibitor is a dsRNA that targetsIDH2, e.g., an IDH2 having a C or a T (or a nucleotide other than G orA) at nucleotide position 516 according to the IDH2 sequence of SEQ IDNO:10.

In an embodiment, the nucleic acid-based inhibitor is a dsRNA thattargets IDH2, e.g., an IDH2 having a G at nucleotide position 514 or a Tat nucleotide position 515 or an A at position 515, according to theIDH2 sequence of SEQ ID NO:10.

In an embodiment, the dsRNA targets an IDH2 having other than A, e.g., aG or a T, at nucleotide position 514, or other than G, e.g., an A or Cor T at position 515 (e.g., a mutant), or other than G, e.g., C or T,and an IDH2 having an A at nucleotide position 514 or a G at nucleotideposition 515 or a G at position 516 (e.g., a wildtype), e.g., bytargeting a region of the IDH2 mRNA that is identical between thewildtype and mutant transcripts. In yet another embodiment, the dsRNAtargets a particular mutant or polymorphism (such as a single nucleotidepolymorphism (SNP)), but not a wildtype allele. In this case, thenucleic acid based inhibitor, e.g., a dsRNA, targets the region of theIDH2 containing the mutation.

In some embodiments, the nucleic acid based inhibitor, e.g., a dsRNA,preferentially or specifically inhibits the product of a mutant IDH2 ascompared to the product of a wildtype IDH2. For example, in oneembodiment, a dsRNA targets a region of an IDH2 mRNA that carries themutation (e.g., an A514G or G515T or a G515U mutation according to SEQID NO:10).

In one embodiment, the nucleic acid-based inhibitor is a dsRNA includinga sense strand and an antisense strand having a primary sequencepresented in Tables 15-23. In another embodiment, the nucleic acid basedinhibitor is an antisense oligonucleotide that includes all or a part ofan antisense primary sequence presented in Tables 15-23 or which targetsthe same or substantially the same region as does a dsRNA from Tables15-23.

In an embodiment, the nucleic acid based inhibitor is delivered to thebrain, e.g., directly to the brain, e.g., by intrathecal orintraventricular delivery. The nucleic acid based inhibitor can also bedelivered from an inplantable device. In an embodiment, the nucleicacid-based inhibitor is delivered by infusion using, e.g., a catheter,and optionally, a pump.

Antisense

Suitable nucleic acid based inhibitors include antisense nucleic acids.While not being bound by theory it is believed that antisense inhibitionis typically based upon hydrogen bonding-based hybridization ofoligonucleotide strands or segments such that at least one strand orsegment is cleaved, degraded, or otherwise rendered inoperable.

An antisense agent can bind IDH1 or IDH2 DNA. In embodiments it inhibitsreplication and transcription. While not being bound by theory it isbelieved that an antisense agent can also function to inhibit target RNAtranslocation, e.g., to a site of protein translation, translation ofprotein from the RNA, splicing of the RNA to yield one or more RNAspecies, and catalytic activity or complex formation involving the RNA.

An antisense agents can have a chemical modification described above asbeing suitable for dsRNA.

Antisense agents can include, for example, from about 8 to about 80nucleobases (i.e., from about 8 to about 80 nucleotides), e.g., about 8to about 50 nucleobases, or about 12 to about 30 nucleobases. Antisensecompounds include ribozymes, external guide sequence (EGS)oligonucleotides (oligozymes), and other short catalytic RNAs orcatalytic oligonucleotides which hybridize to the target nucleic acidand modulate its expression. Anti-sense compounds can include a stretchof at least eight consecutive nucleobases that are complementary to asequence in the target gene. An oligonucleotide need not be 100%complementary to its target nucleic acid sequence to be specificallyhybridizable. An oligonucleotide is specifically hybridizable whenbinding of the oligonucleotide to the target interferes with the normalfunction of the target molecule to cause a loss of utility, and there isa sufficient degree of complementarity to avoid non-specific binding ofthe oligonucleotide to non-target sequences under conditions in whichspecific binding is desired, i.e., under physiological conditions in thecase of in vivo assays or therapeutic treatment or, in the case of invitro assays, under conditions in which the assays are conducted.

Hybridization of antisense oligonucleotides with mRNA (e.g., an mRNAencoding IDH1 or IDH2) can interfere with one or more of the normalfunctions of mRNA. While not being bound by theory it is believed thatthe functions of mRNA to be interfered with include all key functionssuch as, for example, translocation of the RNA to the site of proteintranslation, translation of protein from the RNA, splicing of the RNA toyield one or more mRNA species, and catalytic activity which may beengaged in by the RNA. Binding of specific protein(s) to the RNA mayalso be interfered with by antisense oligonucleotide hybridization tothe RNA.

Exemplary antisense compounds include DNA or RNA sequences thatspecifically hybridize to the target nucleic acid, e.g., the mRNAencoding IDH1 or IDH2. The complementary region can extend for betweenabout 8 to about 80 nucleobases. The compounds can include one or moremodified nucleobases. Modified nucleobases may include, e.g.,5-substituted pyrimidines such as 5-iodouracil, 5-iodocytosine, andC5-propynyl pyrimidines such as C5-propynylcytosine andC5-propynyluracil. Other suitable modified nucleobases includeN⁴—(C₁-C₁₂) alkylaminocytosines and N⁴,N⁴—(C₁-C₁₂)dialkylaminocytosines. Modified nucleobases may also include7-substituted-5-aza-7-deazapurines and 7-substituted-7-deazapurines suchas, for example, 7-iodo-7-deazapurines, 7-cyano-7-deazapurines,7-aminocarbonyl-7-deazapurines. Examples of these include6-amino-7-iodo-7-deazapurines, 6-amino-7-cyano-7-deazapurines,6-amino-7-aminocarbonyl-7-deazapurines,2-amino-6-hydroxy-7-iodo-7-deazapurines,2-amino-6-hydroxy-7-cyano-7-deazapurines, and2-amino-6-hydroxy-7-aminocarbonyl-7-deazapurines. Furthermore,N⁶—(C₁-C₁₂) alkylaminopurines and N⁶,N⁶—(C₁-C₁₂) dialkylaminopurines,including N⁶-methylaminoadenine and N⁶,N⁶-dimethylaminoadenine, are alsosuitable modified nucleobases. Similarly, other 6-substituted purinesincluding, for example, 6-thioguanine may constitute appropriatemodified nucleobases. Other suitable nucleobases include 2-thiouracil,8-bromoadenine, 8-bromoguanine, 2-fluoroadenine, and 2-fluoroguanine.Derivatives of any of the aforementioned modified nucleobases are alsoappropriate. Substituents of any of the preceding compounds may includeC₁-C₃₀ alkyl, C₂-C₃₀ alkenyl, C₂-C₃₀ alkynyl, aryl, aralkyl, heteroaryl,halo, amino, amido, nitro, thio, sulfonyl, carboxyl, alkoxy,alkylcarbonyl, alkoxycarbonyl, and the like.

MicroRNA

In some embodiments, the nucleic acid-based inhibitor suitable fortargeting IDH, e.g., IDH1, is a microRNA (miRNA). A miRNA is a singlestranded RNA that regulates the expression of target mRNAs either bymRNA cleavage, translational repression/inhibition or heterochromaticsilencing. The miRNA is 18 to 25 nucleotides, typically 21 to 23nucleotides in length. In some embodiments, the miRNA includes chemicalmodifications, such as one or more modifications described herein.

In some embodiments, a nucleic acid based inhibitor targeting IDH haspartial complementarity (i.e., less than 100% complementarity) with thetarget IDH, e.g., IDH1 or IDH2, mRNA. For example, partialcomplementarity can include various mismatches or non-base pairednucleotides (e.g., 1, 2, 3, 4, 5 or more mismatches or non-based pairednucleotides, such as nucleotide bulges), which can result in bulges,loops, or overhangs that result between the antisense strand orantisense region of the nucleic acid-based inhibitor and thecorresponding target nucleic acid molecule.

The nucleic acid-based inhibitors described herein, e.g., antisensenucleic acid described herein, can be incorporated into a gene constructto be used as a part of a gene therapy protocol to deliver nucleic acidsthat can be used to express and produce agents within cells. Expressionconstructs of such components may be administered in anybiologically-effective carrier, e.g., any formulation or compositioncapable of effectively delivering the component gene to cells in vivo.Approaches include insertion of the subject gene in viral vectorsincluding recombinant retroviruses, adenovirus, adeno-associated virus,lentivirus, and herpes simplex virus-1, or recombinant bacterial oreukaryotic plasmids. Viral vectors transfect cells directly; plasmid DNAcan be delivered with the help of, for example, cationic liposomes(lipofectin) or derivatized (e.g., antibody conjugated) polylysineconjugates, gramacidin S, artificial viral envelopes or other suchintracellular earners, as well as direct injection of the gene constructor CaPO₄ precipitation carried out in vivo.

In an embodiment, in vivo introduction of nucleic acid into a cellincludes use of a viral vector containing nucleic acid, e.g., a cDNA.Infection of cells with a viral vector has the advantage that a largeproportion of the targeted cells can receive the nucleic acid.Additionally, molecules encoded within the viral vector, e.g., by a cDNAcontained in the viral vector, are expressed efficiently in cells whichhave taken up viral vector nucleic acid.

Retroviral vectors and adeno-associated virus vectors can be used as arecombinant gene delivery system for the transfer of exogenous genes invivo particularly into humans. These vectors provide efficient deliveryof genes into cells, and the transferred nucleic acids are stablyintegrated into the chromosomal DNA of the host. Protocols for producingrecombinant retroviruses and for infecting cells in vitro or in vivowith such viruses can be found in Current Protocols in MolecularBiology, Ausubel, F. M. et al. (eds.) Greene Publishing Associates(1989), Sections 9.10-9.14 and other standard laboratory manuals.Examples of suitable retroviruses include pLJ, pZIP, pWE, and pEM whichare known to those skilled in the art. Examples of suitable packagingvirus lines for preparing both ecotropic and amphotropic retroviralsystems include Crip, Cre, 2, and Am. Retroviruses have been used tointroduce a variety of genes into many different cell types, includingepithelial cells, in vitro and/or in vivo (see, for example, Eglitis etal. (1985) Science 230:1395-1398; Danos and Mulligan (1988) Proc. Natl.Acad. Sci. USA 85:6460-6464; Wilson et al. (1988) Proc. Natl. Acad. Sci.USA 85:3014-3018; Armentano et al. (1990) Proc. Natl. Acad. Sci. USA87:6141-6145; Huber et al. (1991) Proc. Natl. Acad. Sci. USA88:8039-8043; Ferry et al. (1991) Proc. Natl. Acad. Sci. USA88:8377-8381; Chowdhury et al. (1991) Science 254:1802-1805; vanBeusechem et al. (1992) Proc. Natl. Acad. Sci. USA 89:7640-7644; Kay etal. (1992) Human Gene Therapy 3:641-647; Dai et al. (1992) Proc. Natl.Acad. Sci. USA 89:10892-10895; Hwu et al. (1993) J. Immunol.150:4104-4115; U.S. Pat. Nos. 4,868,116 and 4,980,286; PCT Pub. Nos. WO89/07136, WO 89/02468, WO 89/05345, and WO 92/07573).

Another viral gene delivery system utilizes adenovirus-derived vectors.See, for example, Berkner et al. (1988) BioTechniques 6:616; Rosenfeldet al. (1991) Science 252:431-434; and Rosenfeld et al. (1992) Cell68:143-155. Suitable adenoviral vectors derived from the adenovirusstrain Ad type 5 d1324 or other strains of adenovirus (e.g., Ad2, Ad3,Ad7 etc.) are known to those skilled in the art.

Yet another viral vector system useful for delivery of the subject geneis the adeno-associated virus (AAV). See, for example, Flotte et al.(1992) Am. J. Respir. Cell. Mol. Biol. 7:349-356; Samulski et al. (1989)J. Virol. 63:3822-3828; and McLaughlin et al. (1989) J. Virol.62:1963-1973.

Pharmaceutical Compositions

The compositions delineated herein include the compounds delineatedherein, as well as additional therapeutic agents if present, in amountseffective for achieving a modulation of disease or disease symptoms,including those described herein.

The term “pharmaceutically acceptable carrier or adjuvant” refers to acarrier or adjuvant that may be administered to a patient, together witha compound of this invention, and which does not destroy thepharmacological activity thereof and is nontoxic when administered indoses sufficient to deliver a therapeutic amount of the compound.

Pharmaceutically acceptable carriers, adjuvants and vehicles that may beused in the pharmaceutical compositions of this invention include, butare not limited to, ion exchangers, alumina, aluminum stearate,lecithin, self-emulsifying drug delivery systems (SEDDS) such asd-α-tocopherol polyethyleneglycol 1000 succinate, surfactants used inpharmaceutical dosage forms such as Tweens or other similar polymericdelivery matrices, serum proteins, such as human serum albumin, buffersubstances such as phosphates, glycine, sorbic acid, potassium sorbate,partial glyceride mixtures of saturated vegetable fatty acids, water,salts or electrolytes, such as protamine sulfate, disodium hydrogenphosphate, potassium hydrogen phosphate, sodium chloride, zinc salts,colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone,cellulose-based substances, polyethylene glycol, sodiumcarboxymethylcellulose, polyacrylates, waxes,polyethylene-polyoxypropylene-block polymers, polyethylene glycol andwool fat. Cyclodextrins such as α-, β-, and γ-cyclodextrin, orchemically modified derivatives such as hydroxyalkylcyclodextrins,including 2- and 3-hydroxypropyl-β-cyclodextrins, or other solubilizedderivatives may also be advantageously used to enhance delivery ofcompounds of the formulae described herein.

The pharmaceutical compositions containing inhibitors of IDH, e.g.,IDH1, may be administered directly to the central nervous system, suchas into the cerebrospinal fluid or into the brain. Delivery can be, forexample, in a bolus or by continuous pump infusion. In certainembodiments, delivery is by intrathecal delivery or by intraventricularinjection directly into the brain. A catheter and, optionally, a pumpcan be used for delivery. The inhibitors can be delivered in andreleased from an implantable device, e.g., a device that is implanted inassociation with surgical removal of tumor tissue. E.g., for delivery tothe brain, the delivery can be analogous to that with Gliadel, abiopolymer wafer designed to deliver carmustine directly into thesurgical cavity created when a brain tumor is resected. The Gliadelwafer slowly dissolves and delivers carmustine.

The therapeutics disclosed herein, e.g., nucleic acid based inhibitors,e.g. siRNAs can be administered directly to the CNS, e.g., the brain,e.g., using a pump and/or catheter system. In one embodiment, the pumpis implanted under the skin. In an embodiment and a catheter attached toa pump is inserted into the CNS, e.g., into the brain or spine. In oneembodiment, the pump (such as the IsoMed Drug Pump from Medtronic)delivers dosing, e.g, constant dosing, of a nucleic acid basedinhibitor. In an embodiment, the pump is programmable to administervariable or constant doses at predetermined time intervals. For example,the IsoMed Drug pump from Medtronic (or a similar device) can be used toadminister a constant supply of the inhibitor, or the SynchroMedII DrugPump (or a similar device) can be used to administer a variable dosingregime.

Methods and devices described in U.S. Pat. Nos. 7,044,932, 6,620,151,6,283949, and 6,685,452 can be used in methods described herein.

The pharmaceutical compositions of this invention may be administeredorally, parenterally, by inhalation, topically, rectally, nasally,buccally, vaginally or via an implanted reservoir, preferably by oraladministration or administration by injection. The pharmaceuticalcompositions of this invention may contain any conventional non-toxicpharmaceutically-acceptable carriers, adjuvants or vehicles. In somecases, the pH of the formulation may be adjusted with pharmaceuticallyacceptable acids, bases or buffers to enhance the stability of theformulated compound or its delivery form. The term parenteral as usedherein includes subcutaneous, intracutaneous, intravenous,intramuscular, intraarticular, intraarterial, intrasynovial,intrasternal, intrathecal, intralesional and intracranial injection orinfusion techniques.

The pharmaceutical compositions may be in the form of a sterileinjectable preparation, for example, as a sterile injectable aqueous oroleaginous suspension. This suspension may be formulated according totechniques known in the art using suitable dispersing or wetting agents(such as, for example, Tween 80) and suspending agents. The sterileinjectable preparation may also be a sterile injectable solution orsuspension in a non-toxic parenterally acceptable diluent or solvent,for example, as a solution in 1,3-butanediol. Among the acceptablevehicles and solvents that may be employed are mannitol, water, Ringer'ssolution and isotonic sodium chloride solution. In addition, sterile,fixed oils are conventionally employed as a solvent or suspendingmedium. For this purpose, any bland fixed oil may be employed includingsynthetic mono- or diglycerides. Fatty acids, such as oleic acid and itsglyceride derivatives are useful in the preparation of injectables, asare natural pharmaceutically-acceptable oils, such as olive oil orcastor oil, especially in their polyoxyethylated versions. These oilsolutions or suspensions may also contain a long-chain alcohol diluentor dispersant, or carboxymethyl cellulose or similar dispersing agentswhich are commonly used in the formulation of pharmaceuticallyacceptable dosage forms such as emulsions and or suspensions. Othercommonly used surfactants such as Tweens or Spans and/or other similaremulsifying agents or bioavailability enhancers which are commonly usedin the manufacture of pharmaceutically acceptable solid, liquid, orother dosage forms may also be used for the purposes of formulation.

The pharmaceutical compositions of this invention may be orallyadministered in any orally acceptable dosage form including, but notlimited to, capsules, tablets, emulsions and aqueous suspensions,dispersions and solutions. In the case of tablets for oral use, carrierswhich are commonly used include lactose and corn starch. Lubricatingagents, such as magnesium stearate, are also typically added. For oraladministration in a capsule form, useful diluents include lactose anddried corn starch. When aqueous suspensions and/or emulsions areadministered orally, the active ingredient may be suspended or dissolvedin an oily phase is combined with emulsifying and/or suspending agents.If desired, certain sweetening and/or flavoring and/or coloring agentsmay be added.

The pharmaceutical compositions of this invention may also beadministered in the form of suppositories for rectal administration.These compositions can be prepared by mixing a compound of thisinvention with a suitable non-irritating excipient which is solid atroom temperature but liquid at the rectal temperature and therefore willmelt in the rectum to release the active components. Such materialsinclude, but are not limited to, cocoa butter, beeswax and polyethyleneglycols.

Topical administration of the pharmaceutical compositions of thisinvention is useful when the desired treatment involves areas or organsreadily accessible by topical application. For application topically tothe skin, the pharmaceutical composition should be formulated with asuitable ointment containing the active components suspended ordissolved in a carrier. Carriers for topical administration of thecompounds of this invention include, but are not limited to, mineraloil, liquid petroleum, white petroleum, propylene glycol,polyoxyethylene polyoxypropylene compound, emulsifying wax and water.Alternatively, the pharmaceutical composition can be formulated with asuitable lotion or cream containing the active compound suspended ordissolved in a carrier with suitable emulsifying agents. Suitablecarriers include, but are not limited to, mineral oil, sorbitanmonostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol,2-octyldodecanol, benzyl alcohol and water. The pharmaceuticalcompositions of this invention may also be topically applied to thelower intestinal tract by rectal suppository formulation or in asuitable enema formulation. Topically-transdermal patches are alsoincluded in this invention.

The pharmaceutical compositions of this invention may be administered bynasal aerosol or inhalation. Such compositions are prepared according totechniques well-known in the art of pharmaceutical formulation and maybe prepared as solutions in saline, employing benzyl alcohol or othersuitable preservatives, absorption promoters to enhance bioavailability,fluorocarbons, and/or other solubilizing or dispersing agents known inthe art.

When the compositions of this invention comprise a combination of acompound of the formulae described herein and one or more additionaltherapeutic or prophylactic agents, both the compound and the additionalagent should be present at dosage levels of between about 1 to 100%, andmore preferably between about 5 to 95% of the dosage normallyadministered in a monotherapy regimen. The additional agents may beadministered separately, as part of a multiple dose regimen, from thecompounds of this invention. Alternatively, those agents may be part ofa single dosage form, mixed together with the compounds of thisinvention in a single composition.

The compounds described herein can, for example, be administered byinjection, intravenously, intraarterially, subdermally,intraperitoneally, intramuscularly, or subcutaneously; or orally,buccally, nasally, transmucosally, topically, in an ophthalmicpreparation, or by inhalation, with a dosage ranging from about 0.02 toabout 100 mg/kg of body weight, alternatively dosages between 1 mg and1000 mg/dose, every 4 to 120 hours, or according to the requirements ofthe particular drug. The methods herein contemplate administration of aneffective amount of compound or compound composition to achieve thedesired or stated effect. Typically, the pharmaceutical compositions ofthis invention will be administered from about 1 to about 6 times perday or alternatively, as a continuous infusion. Such administration canbe used as a chronic or acute therapy. The amount of active ingredientthat may be combined with the carrier materials to produce a singledosage form will vary depending upon the host treated and the particularmode of administration. A typical preparation will contain from about 5%to about 95% active compound (w/w). Alternatively, such preparationscontain from about 20% to about 80% active compound.

Lower or higher doses than those recited above may be required. Specificdosage and treatment regimens for any particular patient will dependupon a variety of factors, including the activity of the specificcompound employed, the age, body weight, general health status, sex,diet, time of administration, rate of excretion, drug combination, theseverity and course of the disease, condition or symptoms, the patient'sdisposition to the disease, condition or symptoms, and the judgment ofthe treating physician.

Upon improvement of a patient's condition, a maintenance dose of acompound, composition or combination of this invention may beadministered, if necessary. Subsequently, the dosage or frequency ofadministration, or both, may be reduced, as a function of the symptoms,to a level at which the improved condition is retained when the symptomshave been alleviated to the desired level. Patients may, however,require intermittent treatment on a long-term basis upon any recurrenceof disease symptoms.

Kits

A compound described herein can be provided in a kit.

In an embodiment the kit includes (a) a compound described herein, e.g.,a composition that includes a compound described herein (wherein, e.g.,the compound can be an inhibitor described herein), and, optionally (b)informational material. The informational material can be descriptive,instructional, marketing or other material that relates to the methodsdescribed herein and/or the use of a compound described herein for themethods described herein.

In an embodiment the kit provides materials for evaluating a subject.The evaluation can be, e.g., for: identifying a subject having unwantedlevels (e.g., higher than present in normal or wildtype cells) of any of2HG, 2HG neoactivity, or mutant IDH1 or IDH2 protein having 2HGneoactivity (or corresponding RNA), or having a somatic mutation in IDH1or IDH2 characterized by 2HG neoactivity; diagnosing, prognosing, orstaging, a subject, e.g., on the basis of having increased levels of2HG, 2HG neoactivity, or mutant IDH1 or IDH2 protein having 2HGneoactivity (or corresponding RNA), or having a somatic mutation in IDH1or IDH2 characterized by 2HG neoactivity; selecting a treatment for, orevaluating the efficacy of, a treatment, e.g., on the basis of thesubject having increased levels of 2HG, 2HG neoactivity, or mutant IDH1or IDH2 protein having 2HG neoactivity (or corresponding RNA), or havinga somatic mutation in IDH1 or IDH2 characterized by 2HG neoactivity. Thekit can include one or more reagent useful in the evaluation, e.g.,reagents mentioned elsewhere herein. A detection reagent, e.g., anantibody or other specific binding reagent can be included. Standards orreference samples, e.g., a positive or negative control standard can beincluded. E.g., if the evaluation is based on the presence of 2HG thekit can include a reagent, e.g, a positive or negative control standardsfor an assay, e.g., a LC-MS assay.

If the evaluation is based on the presence of 2HG neoactivity, the kitcan include a reagent, e.g., one or more of those mentioned elsewhereherein, for assaying 2HG neoactivity. If the evaluation is based onsequencing, the kit can include primers or other materials useful forsequencing the relevant nucleic acids for identifying an IHD, e.g., IDH1or IDH2, neoactive mutant. E.g., the kit can contain a reagent thatprovides for interrogation of the indentity, i.e., sequencing of,residue 132 of IDH1 to determine if a neoactive mutant is present. Thekit can include nucleic acids, e.g., an oligomer, e.g., primers, whichallow sequencing of of the nucleotides that encode residue 132 of IDH1.In an embodiment the kit includes a nucleic acid whose hybridization, orability to be amplified, is dependent on the indentity of residue 132 ofIDH1. In other embodiments the kit includes a reagent, e.g., an antibodyor other specific binding molecule that can identify the presence of aneoactive mutant, e.g., a protein encoded by a neoactive mutant at 132of IDH1. As described below, a kit can also include buffers, solvents,and information related to the evaluation.

In one embodiment, the informational material can include informationabout production of the compound, molecular weight of the compound,concentration, date of expiration, batch or production site information,and so forth. In one embodiment, the informational material relates tomethods for administering the compound.

In one embodiment, the informational material can include instructionsto administer a compound described herein in a suitable manner toperform the methods described herein, e.g., in a suitable dose, dosageform, or mode of administration (e.g., a dose, dosage form, or mode ofadministration described herein). In another embodiment, theinformational material can include instructions to administer a compounddescribed herein to a suitable subject, e.g., a human, e.g., a humanhaving or at risk for a disorder described herein.

The informational material of the kits is not limited in its form. Inmany cases, the informational material, e.g., instructions, is providedin printed matter, e.g., a printed text, drawing, and/or photograph,e.g., a label or printed sheet. However, the informational material canalso be provided in other formats, such as Braille, computer readablematerial, video recording, or audio recording. In another embodiment,the informational material of the kit is contact information, e.g., aphysical address, email address, website, or telephone number, where auser of the kit can obtain substantive information about a compounddescribed herein and/or its use in the methods described herein. Ofcourse, the informational material can also be provided in anycombination of formats.

In addition to a compound described herein, the composition of the kitcan include other ingredients, such as a solvent or buffer, astabilizer, a preservative, a flavoring agent (e.g., a bitter antagonistor a sweetener), a fragrance or other cosmetic ingredient, and/or asecond agent for treating a condition or disorder described herein.Alternatively, the other ingredients can be included in the kit, but indifferent compositions or containers than a compound described herein.In such embodiments, the kit can include instructions for admixing acompound described herein and the other ingredients, or for using acompound described herein together with the other ingredients.

A compound described herein can be provided in any form, e.g., liquid,dried or lyophilized form. It is preferred that a compound describedherein be substantially pure and/or sterile. When a compound describedherein is provided in a liquid solution, the liquid solution preferablyis an aqueous solution, with a sterile aqueous solution being preferred.When a compound described herein is provided as a dried form,reconstitution generally is by the addition of a suitable solvent. Thesolvent, e.g., sterile water or buffer, can optionally be provided inthe kit.

The kit can include one or more containers for the compositioncontaining a compound described herein. In some embodiments, the kitcontains separate containers, dividers or compartments for thecomposition and informational material. For example, the composition canbe contained in a bottle, vial, or syringe, and the informationalmaterial can be contained in a plastic sleeve or packet. In otherembodiments, the separate elements of the kit are contained within asingle, undivided container. For example, the composition is containedin a bottle, vial or syringe that has attached thereto the informationalmaterial in the form of a label. In some embodiments, the kit includes aplurality (e.g., a pack) of individual containers, each containing oneor more unit dosage forms (e.g., a dosage form described herein) of acompound described herein. For example, the kit includes a plurality ofsyringes, ampules, foil packets, or blister packs, each containing asingle unit dose of a compound described herein. The containers of thekits can be air tight, waterproof (e.g., impermeable to changes inmoisture or evaporation), and/or light-tight.

The kit optionally includes a device suitable for administration of thecomposition, e.g., a syringe, inhalant, pipette, forceps, measuredspoon, dropper (e.g., eye dropper), swab (e.g., a cotton swab or woodenswab), or any such delivery device. In an embodiment, the device is amedical implant device, e.g., packaged for surgical insertion.

Combination Therapies

In some embodiments, a compound or composition described herein, isadministered together with an additional cancer treatment. Exemplarycancer treatments include, for example: surgery, chemotherapy, targetedtherapies such as antibody therapies, immunotherapy, and hormonaltherapy. Examples of each of these treatments are provided below.

Chemotherapy

In some embodiments, a compound or composition described herein, isadministered with a chemotherapy. Chemotherapy is the treatment ofcancer with drugs that can destroy cancer cells. “Chemotherapy” usuallyrefers to cytotoxic drugs which affect rapidly dividing cells ingeneral, in contrast with targeted therapy. Chemotherapy drugs interferewith cell division in various possible ways, e.g., with the duplicationof DNA or the separation of newly formed chromosomes. Most forms ofchemotherapy target all rapidly dividing cells and are not specific forcancer cells, although some degree of specificity may come from theinability of many cancer cells to repair DNA damage, while normal cellsgenerally can.

Examples of chemotherapeutic agents used in cancer therapy include, forexample, antimetabolites (e.g., folic acid, purine, and pyrimidinederivatives) and alkylating agents (e.g., nitrogen mustards,nitrosoureas, platinum, alkyl sulfonates, hydrazines, triazenes,aziridines, spindle poison, cytotoxic agents, toposimerase inhibitorsand others). Exemplary agents include Aclarubicin, Actinomycin,Alitretinon, Altretamine, Aminopterin, Aminolevulinic acid, Amrubicin,Amsacrine, Anagrelide, Arsenic trioxide, Asparaginase, Atrasentan,Belotecan, Bexarotene, endamustine, Bleomycin, Bortezomib, Busulfan,Camptothecin, Capecitabine, Carboplatin, Carboquone, Carmofur,Carmustine, Celecoxib, Chlorambucil, Chlormethine, Cisplatin,Cladribine, Clofarabine, Crisantaspase, Cyclophosphamide, Cytarabine,Dacarbazine, Dactinomycin, Daunorubicin, Decitabine, Demecolcine,Docetaxel, Doxorubicin, Efaproxiral, Elesclomol, Elsamitrucin,Enocitabine, Epirubicin, Estramustine, Etoglucid, Etoposide,Floxuridine, Fludarabine, Fluorouracil (5FU), Fotemustine, Gemcitabine,Gliadel implants, Hydroxycarbamide, Hydroxyurea, Idarubicin, Ifosfamide,Irinotecan, Irofulven, Ixabepilone, Larotaxel, Leucovorin, Liposomaldoxorubicin, Liposomal daunorubicin, Lonidamine, Lomustine, Lucanthone,Mannosulfan, Masoprocol, Melphalan, Mercaptopurine, Mesna, Methotrexate,Methyl aminolevulinate, Mitobronitol, Mitoguazone, Mitotane, Mitomycin,Mitoxantrone, Nedaplatin, Nimustine, Oblimersen, Omacetaxine, Ortataxel,Oxaliplatin, Paclitaxel, Pegaspargase, Pemetrexed, Pentostatin,Pirarubicin, Pixantrone, Plicamycin, Porfimer sodium, Prednimustine,Procarbazine, Raltitrexed, Ranimustine, Rubitecan, Sapacitabine,Semustine, Sitimagene ceradenovec, Strataplatin, Streptozocin,Talaporfin, Tegafur-uracil, Temoporfin, Temozolomide, Teniposide,Tesetaxel, Testolactone, Tetranitrate, Thiotepa, Tiazofurine,Tioguanine, Tipifarnib, Topotecan, Trabectedin, Triaziquone,Triethylenemelamine, Triplatin, Tretinoin, Treosulfan, Trofosfamide,Uramustine, Valrubicin, Verteporfin, Vinblastine, Vincristine,Vindesine, Vinflunine, Vinorelbine, Vorinostat, Zorubicin, and othercytostatic or cytotoxic agents described herein.

Because some drugs work better together than alone, two or more drugsare often given at the same time. Often, two or more chemotherapy agentsare used as combination chemotherapy. In some embodiments, thechemotherapy agents (including combination chemotherapy) can be used incombination with a compound described herein, e.g., phenformin.

Targeted Therapy

In some embodiments, a compound or composition described herein, isadministered with a targeted therapy. Targeted therapy constitutes theuse of agents specific for the deregulated proteins of cancer cells.Small molecule targeted therapy drugs are generally inhibitors ofenzymatic domains on mutated, overexpressed, or otherwise criticalproteins within the cancer cell. Prominent examples are the tyrosinekinase inhibitors such as Axitinib, Bosutinib, Cediranib, desatinib,erlotinib, imatinib, gefitinib, lapatinib, Lestaurtinib, Nilotinib,Semaxanib, Sorafenib, Sunitinib, and Vandetanib, and alsocyclin-depdendent kinase inhibitors such as Alvocidib and Seliciclib.Monoclonal antibody therapy is another strategy in which the therapeuticagent is an antibody which specifically binds to a protein on thesurface of the cancer cells. Examples include the anti-HER2/neu antibodytrastuzumab (HERCEPTIN®) typically used in breast cancer, and theanti-CD20 antibody rituximab and Tositumomab typically used in a varietyof B-cell malignancies. Other exemplary antibodies include Cetuximab,Panitumumab, Trastuzumab, Alemtuzumab, Bevacizumab, Edrecolomab, andGemtuzumab. Exemplary fusion proteins include Aflibercept and Denileukindiftitox. In some embodiments, the targeted therapy can be used incombination with a compound described herein, e.g., a biguanide such asmetformin or phenformin, preferably phenformin.

Targeted therapy can also involve small peptides as “homing devices”which can bind to cell surface receptors or affected extracellularmatrix surrounding the tumor. Radionuclides which are attached to thesepeptides (e.g., RGDs) eventually kill the cancer cell if the nuclidedecays in the vicinity of the cell. An example of such therapy includesBEXXAR®.

Immunotherapy

In some embodiments, a compound or composition described herein, isadministered with an immunotherapy. Cancer immunotherapy refers to adiverse set of therapeutic strategies designed to induce the patient'sown immune system to fight the tumor. Contemporary methods forgenerating an immune response against tumors include intravesicular BCGimmunotherapy for superficial bladder cancer, and use of interferons andother cytokines to induce an immune response in renal cell carcinoma andmelanoma patients.

Allogeneic hematopoietic stem cell transplantation can be considered aform of immunotherapy, since the donor's immune cells will often attackthe tumor in a graft-versus-tumor effect. In some embodiments, theimmunotherapy agents can be used in combination with a compound orcomposition described herein.

Hormonal Therapy

In some embodiments, a compound or composition described herein, isadministered with a hormonal therapy. The growth of some cancers can beinhibited by providing or blocking certain hormones. Common examples ofhormone-sensitive tumors include certain types of breast and prostatecancers. Removing or blocking estrogen or testosterone is often animportant additional treatment. In certain cancers, administration ofhormone agonists, such as progestogens may be therapeuticallybeneficial. In some embodiments, the hormonal therapy agents can be usedin combination with a compound or a composition described herein.

In some embodiments, a compound or composition described herein, isadministered together with an additional cancer treatment (e.g.,surgical removal), in treating cancer in nervous system, e.g., cancer incentral nervous system, e.g., brain tumor, e.g., glioma, e.g.,glioblastoma multiforme (GBM).

Several studies have suggested that more than 25% of glioblastomapatients obtain a significant survival benefit from adjuvantchemotherapy. Meta-analyses have suggested that adjuvant chemotherapyresults in a 6-10% increase in 1-year survival rate.

Temozolomide is an orally active alkylating agent that is used forpersons newly diagnosed with glioblastoma multiforme. It was approved bythe United States Food and Drug Administration (FDA) in March 2005.Studies have shown that the drug was well tolerated and provided asurvival benefit. Adjuvant and concomitant temozolomide with radiationwas associated with significant improvements in median progression-freesurvival over radiation alone (6.9 vs 5 mo), overall survival (14.6 vs12.1 mo), and the likelihood of being alive in 2 years (26% vs 10%).

Nitrosoureas: BCNU (carmustine)-polymer wafers (Gliadel) were approvedby the FDA in 2002. Though Gliadel wafers are used by some for initialtreatment, they have shown only a modest increase in median survivalover placebo (13.8 vs. 11.6 months) in the largest such phase III trial,and are associated with increased rates of CSF leak and increasedintracranial pressure secondary to edema and mass effect.

MGMT is a DNA repair enzyme that contributes to temozolomide resistance.Methylation of the MGMT promoter, found in approximately 45% ofglioblastoma multiformes, results in an epigenetic silencing of thegene, decreasing the tumor cell's capacity for DNA repair and increasingsusceptibility to temozolomide.

When patients with and without MGMT promoter methylation were treatedwith temozolomide, the groups had median survivals of 21.7 versus 12.7months, and 2-year survival rates of 46% versus 13.8%, respectively.

Though temozolomide is currently a first-line agent in the treatment ofglioblastoma multiforme, unfavorable MGMT methylation status could helpselect patients appropriate for future therapeutic investigations.

O6-benzylguanine and other inhibitors of MGMT as well as RNAinterference-mediated silencing of MGMT offer promising avenues toincrease the effectiveness of temozolomide and other alkylatingantineoplastics, and such agents are under active study.

Carmustine (BCNU) and cis-platinum (cisplatin) have been the primarychemotherapeutic agents used against malignant gliomas. All agents inuse have no greater than a 30-40% response rate, and most fall into therange of 10-20%.

Data from the University of California at San Francisco indicate that,for the treatment of glioblastomas, surgery followed by radiationtherapy leads to 1-, 3-, and 5-year survival rates of 44%, 6%, and 0%,respectively. By comparison, surgery followed by radiation andchemotherapy using nitrosourea-based regimens resulted in 1-, 3-, and5-year survival rates of 46%, 18%, and 18%, respectively.

A major hindrance to the use of chemotherapeutic agents for brain tumorsis the fact that the blood-brain barrier (BBB) effectively excludes manyagents from the CNS. For this reason, novel methods of intracranial drugdelivery are being developed to deliver higher concentrations ofchemotherapeutic agents to the tumor cells while avoiding the adversesystemic effects of these medications.

Pressure-driven infusion of chemotherapeutic agents through anintracranial catheter, also known as convection-enhanced delivery (CED),has the advantage of delivering drugs along a pressure gradient ratherthan by simple diffusion. CED has shown promising results in animalmodels with agents including BCNU and topotecan.

Initial attempts investigated the delivery of chemotherapeutic agentsvia an intraarterial route rather than intravenously. Unfortunately, nosurvival advantage was observed.

Chemotherapy for recurrent glioblastoma multiforme provides modest, ifany, benefit, and several classes of agents are used. Carmustine wafersincreased 6-month survival from 36% to 56% over placebo in onerandomized study of 222 patients, though there was a significantassociation between the treatment group and serious intracranialinfections.

Genotyping of brain tumors may have applications in stratifying patientsfor clinical trials of various novel therapies.

The anti-angiogenic agent bevacizumab, when used with irinotecanimproved 6-month survival in recurrent glioma patients to 46% comparedwith 21% in patients treated with temozolomide. This bevacizumab andirinotecan combination for recurrent glioblastoma multiforme has beenshown to improve survival over bevacizumab alone. Anti-angiogenic agentsalso decrease peritumoral edema, potentially reducing the necessarycorticosteroid dose.

Some glioblastomas responds to gefitinib or erlotinib (tyrosine kinaseinhibitors). The simultaneous presence in glioblastoma cells of mutantEGFR (EGFRviii) and PTEN was associated with responsiveness to tyrosinekinase inhibitors, whereas increased p-akt predicts a decreased effect.Other targets include PDGFR, VEGFR, mTOR, farnesyltransferase, and PI3K.

Other possible therapy modalities include imatinib, gene therapy,peptide and dendritic cell vaccines, synthetic chlorotoxins, andradiolabeled drugs and antibodies.

Patient Selection/Monitoring

Described herein are methods of treating a cell proliferation-relateddisorder, e.g., cancer, in a subject and methods of identifying asubject for a treatment described herein. Also described herein aremethods of predicting a subject who is at risk of developing cancer(e.g., a cancer associate with a mutation in an enzyme (e.g., an enzymein the metabolic pathway such as IDH1 and/or IDH2)). The cancer isgenerally characterized by the presence of a neoactivity, such as a gainof function in one or more mutant enzymes (e.g., an enzyme in themetabolic pathway leading to fatty acid biosynthesis, glycolysis,glutaminolysis, the pentose phosphate shunt, the nucleotide biosyntheticpathway, or the fatty acid biosynthetic pathway, e.g., IDH1 or IDH2).The subject can be selected on the basis of the subject having a mutantgene having a neoactivity, e.g., a neoactivity described herein. As usedherein, “select” means selecting in whole or part on said basis.

In some embodiments, a subject is selected for treatment with a compounddescribed herein based on a determination that the subject has a mutantenzyme described herein (e.g., an enzyme in the metabolic pathway, e.g.,a metabolic pathway leading to fatty acid biosynthesis, glycolysis,glutaminolysis, the pentose phosphate shunt, the nucleotide biosyntheticpathway, or the fatty acid biosynthetic pathway, e.g., IDH1 or IDH2). Insome embodiments, the mutant enzyme has a neoactivity and the patient isselected on that basis. The neoactivity of the enzyme can be identified,for example, by evaluating the subject or sample (e.g., tissue or bodilyfluid) therefrom, for the presence or amount of a substrate, cofactorand/or product of the enzyme. The presence and/or amount of substrate,cofactor and/or product can correspond to the wild-type/non-mutantactivity or can correspond to the neoactivity of the enzyme. Exemplarybodily fluid that can be used to identify (e.g., evaluate) theneoactivity of the enzyme include amniotic fluid surrounding a fetus,aqueous humour, blood (e.g., blood plasma), Cerebrospinal fluid,cerumen, chyme, Cowper's fluid, female ejaculate, interstitial fluid,lymph, breast milk, mucus (e.g., nasal drainage or phlegm), pleuralfluid, pus, saliva, sebum, semen, serum, sweat, tears, urine, vaginalsecretion, or vomit.

In some embodiments, a subject can be evaluated for neoactivity of anenzyme using magnetic resonance. For example, where the mutant enzyme isIDH1 or IDH2 and the neoactivity is conversion of α-ketoglutarate to2-hydroxyglutarate, the subject can be evaluated for the presence ofand/or an elevated amount of 2-hydroxyglutarate, e.g.,R-2-hydroxyglutarate relative to the amount of 2-hydroxyglutarate, e.g.,R-2-hydroxyglutarate present in a subject who does not have a mutationin IDH1 or IDH2 having the above neoactivity. In some embodiments,neoactivity of IDH1 or IDH2 can be determined by the presence orelevated amount of a peak corresponding to 2-hydroxyglutarate, e.g.,R-2-hydroxyglutarate as determined by magnetic resonance. For example, asubject can be evaluated for the presence and/or strength of a signal atabout 2.5 ppm to determine the presence and/or amount of2-hydroxyglutarate, e.g., R-2-hydroxyglutarate in the subject. This canbe correlated to and/or predictive of a neoactivity described herein forthe mutant enzyme IDH. Similarly, the presence, strength and/or absenceof a signal at about 2.5 ppm could be predictive of a response totreatment and thereby used as a noninvasive biomarker for clinicalresponse.

Neoactivity of a mutant enzyme such as IDH can also be evaluated usingother techniques known to one skilled in the art. For example, thepresence or amount of a labeled substrate, cofactor, and/or reactionproduct can be measured such as a ¹³C or ¹⁴C labeled substrate,cofactor, and/or reaction product. The neoactivity can be evaluated byevaluating the forward reaction of the wild-type/non mutant enzyme (suchas the oxidative decarboxylation of ioscitrate to α-ketoglutarate in amutant IDH1 or IDH2 enzyme, specifically a mutant IDH1 enzyme) and/orthe reaction corresponding to the neoactivity (e.g., the conversion ofα-ketoglutarate to 2-hydroxyglutarate, e.g., R-2-hydroxyglutarate in amutant IDH1 or IDH2 enzyme, specifically a mutant IDH1 enzyme).

Disorders

The IDH-related methods disclosed herein, e.g., methods of evaluating ortreating subjects, are directed to subjects having a cellproliferation-related disorder characterized by an IDH mutant, e.g., anIDH1 or IDH2, mutant having neoactivity, e.g., 2HG neoactivity. Examplesof some of the disorders below have been shown to be characterized by anIDH1 or IDH2 mutation. Others can be analyzed, e.g., by sequencing cellsamples to determine the presence of a somatic mutation at amino acid132 of IDH1 or at amino acid 172 of IDH2. Without being bound by theoryit is expected that a portion of the tumors of given type of cancer willhave an IDH, e.g., IDH1 or IDH2, mutant having 2HG neoactivity.

The disclosed methods are useful in evaluating or treating proliferativedisorders, e.g. evaluating or treating solid tumors, soft tissue tumors,and metastases thereof wherein the solid tumor, soft tissue tumor ormetastases thereof is a cancer described herein. Exemplary solid tumorsinclude malignancies (e.g., sarcomas, adenocarcinomas, and carcinomas)of the various organ systems, such as those of brain, lung, breast,lymphoid, gastrointestinal (e.g., colon), and genitourinary (e.g.,renal, urothelial, or testicular tumors) tracts, pharynx, prostate, andovary. Exemplary adenocarcinomas include colorectal cancers, renal-cellcarcinoma, liver cancer, non-small cell carcinoma of the lung, andcancer of the small intestine. The disclosed methods are also useful inevaluating or treating non-solid cancers.

The methods described herein can be used with any cancer, for examplethose described by the National Cancer Institute. A cancer can beevaluated to determine whether it is using a method described herein.Exemplary cancers described by the National Cancer Institute include:Acute Lymphoblastic Leukemia, Adult; Acute Lymphoblastic Leukemia,Childhood; Acute Myeloid Leukemia, Adult; Adrenocortical Carcinoma;Adrenocortical Carcinoma, Childhood; AIDS-Related Lymphoma; AIDS-RelatedMalignancies; Anal Cancer; Astrocytoma, Childhood Cerebellar;Astrocytoma, Childhood Cerebral; Bile Duct Cancer, Extrahepatic; BladderCancer; Bladder Cancer, Childhood; Bone Cancer, Osteosarcoma/MalignantFibrous Histiocytoma; Brain Stem Glioma, Childhood; Brain Tumor, Adult;Brain Tumor, Brain Stem Glioma, Childhood; Brain Tumor, CerebellarAstrocytoma, Childhood; Brain Tumor, Cerebral Astrocytoma/MalignantGlioma, Childhood; Brain Tumor, Ependymoma, Childhood; Brain Tumor,Medulloblastoma, Childhood; Brain Tumor, Supratentorial PrimitiveNeuroectodermal Tumors, Childhood; Brain Tumor, Visual Pathway andHypothalamic Glioma, Childhood; Brain Tumor, Childhood (Other); BreastCancer; Breast Cancer and Pregnancy; Breast Cancer, Childhood; BreastCancer, Male; Bronchial Adenomas/Carcinoids, Childhood; Carcinoid Tumor,Childhood; Carcinoid Tumor, Gastrointestinal; Carcinoma, Adrenocortical;Carcinoma, Islet Cell; Carcinoma of Unknown Primaiy; Central NervousSystem Lymphoma, Primary; Cerebellar Astrocytoma, Childhood; CerebralAstrocytoma/Malignant Glioma, Childhood; Cervical Cancer; ChildhoodCancers; Chronic Lymphocytic Leukemia; Chronic Myelogenous Leukemia;Chronic Myeloproliferative Disorders; Clear Cell Sarcoma of TendonSheaths; Colon Cancer; Colorectal Cancer, Childhood; Cutaneous T-CellLymphoma; Endometrial Cancer; Ependymoma, Childhood; Epithelial Cancer,Ovarian; Esophageal Cancer; Esophageal Cancer, Childhood; Ewing's Familyof Tumors; Extracranial Germ Cell Tumor, Childhood; Extragonadal GermCell Tumor; Extrahepatic Bile Duct Cancer; Eye Cancer, IntraocularMelanoma; Eye Cancer, Retinoblastoma; Gallbladder Cancer; Gastric(Stomach) Cancer; Gastric (Stomach) Cancer, Childhood; GastrointestinalCarcinoid Tumor; Germ Cell Tumor, Extracranial, Childhood; Germ CellTumor, Extragonadal; Germ Cell Tumor, Ovarian; Gestational TrophoblasticTumor; Glioma, Childhood Brain Stem; Glioma, Childhood Visual Pathwayand Hypothalamic; Hairy Cell Leukemia; Head and Neck Cancer;Hepatocellular (Liver) Cancer, Adult (Primary); Hepatocellular (Liver)Cancer, Childhood (Primary); Hodgkin's Lymphoma, Adult; Hodgkin'sLymphoma, Childhood; Hodgkin's Lymphoma During Pregnancy; HypopharyngealCancer; Hypothalamic and Visual Pathway Glioma, Childhood; IntraocularMelanoma; Islet Cell Carcinoma (Endocrine Pancreas); Kaposi's Sarcoma;Kidney Cancer; Laryngeal Cancer; Laryngeal Cancer, Childhood; Leukemia,Acute Lymphoblastic, Adult; Leukemia, Acute Lymphoblastic, Childhood;Leukemia, Acute Myeloid, Adult; Leukemia, Acute Myeloid, Childhood;Leukemia, Chronic Lymphocytic; Leukemia, Chronic Myelogenous; Leukemia,Hairy Cell; Lip and Oral Cavity Cancer; Liver Cancer, Adult (Primary);Liver Cancer, Childhood (Primary); Lung Cancer, Non-Small Cell; LungCancer, Small Cell; Lymphoblastic Leukemia, Adult Acute; LymphoblasticLeukemia, Childhood Acute; Lymphocytic Leukemia, Chronic; Lymphoma,AIDS-Related; Lymphoma, Central Nervous System (Primary); Lymphoma,Cutaneous T-Cell; Lymphoma, Hodgkin's, Adult; Lymphoma, Hodgkin's,Childhood; Lymphoma, Hodgkin's During Pregnancy; Lymphoma,Non-Hodgkin's, Adult; Lymphoma, Non-Hodgkin's, Childhood; Lymphoma,Non-Hodgkin's During Pregnancy; Lymphoma, Primary Central NervousSystem; Macroglobulinemia, Waldenstrom's; Male Breast Cancer; MalignantMesothelioma, Adult; Malignant Mesothelioma, Childhood; MalignantThymoma; Medulloblastoma, Childhood; Melanoma; Melanoma, Intraocular;Merkel Cell Carcinoma; Mesothelioma, Malignant; Metastatic Squamous NeckCancer with Occult Primary; Multiple Endocrine Neoplasia Syndrome,Childhood; Multiple Myeloma/Plasma Cell Neoplasm; Mycosis Fungoides;Myelodysplastic Syndromes; Myelogenous Leukemia, Chronic; MyeloidLeukemia, Childhood Acute; Myeloma, Multiple; MyeloproliferativeDisorders, Chronic; Nasal Cavity and Paranasal Sinus Cancer;Nasopharyngeal Cancer; Nasopharyngeal Cancer, Childhood; Neuroblastoma;Non-Hodgkin's Lymphoma, Adult; Non-Hodgkin's Lymphoma, Childhood;Non-Hodgkin's Lymphoma During Pregnancy; Non-Small Cell Lung Cancer;Oral Cancer, Childhood; Oral Cavity and Lip Cancer; OropharyngealCancer; Osteosarcoma/Malignant Fibrous Histiocytoma of Bone; OvarianCancer, Childhood; Ovarian Epithelial Cancer; Ovarian Germ Cell Tumor;Ovarian Low Malignant Potential Tumor; Pancreatic Cancer; PancreaticCancer, Childhood; Pancreatic Cancer, Islet Cell; Paranasal Sinus andNasal Cavity Cancer; Parathyroid Cancer; Penile Cancer;Pheochromocytoma; Pineal and Supratentorial Primitive NeuroectodermalTumors, Childhood; Pituitary Tumor; Plasma Cell Neoplasm/MultipleMyeloma; Pleuropulmonary Blastoma; Pregnancy and Breast Cancer;Pregnancy and Hodgkin's Lymphoma; Pregnancy and Non-Hodgkin's Lymphoma;Primary Central Nervous System Lymphoma; Primary Liver Cancer, Adult;Primary Liver Cancer, Childhood; Prostate Cancer; Rectal Cancer; RenalCell (Kidney) Cancer; Renal Cell Cancer, Childhood; Renal Pelvis andUreter, Transitional Cell Cancer; Retinoblastoma; Rhabdomyosarcoma,Childhood; Salivary Gland Cancer; Salivary Gland Cancer, Childhood;Sarcoma, Ewing's Family of Tumors; Sarcoma, Kaposi's; Sarcoma(Osteosarcoma)/Malignant Fibrous Histiocytoma of Bone; Sarcoma,Rhabdomyosarcoma, Childhood; Sarcoma, Soft Tissue, Adult; Sarcoma, SoftTissue, Childhood; Sezary Syndrome; Skin Cancer; Skin Cancer, Childhood;Skin Cancer (Melanoma); Skin Carcinoma, Merkel Cell; Small Cell LungCancer; Small Intestine Cancer; Soft Tissue Sarcoma, Adult; Soft TissueSarcoma, Childhood; Squamous Neck Cancer with Occult Primary,Metastatic; Stomach (Gastric) Cancer; Stomach (Gastric) Cancer,Childhood; Supratentorial Primitive Neuroectodermal Tumors, Childhood;T-Cell Lymphoma, Cutaneous; Testicular Cancer; Thymoma, Childhood;Thymoma, Malignant; Thyroid Cancer; Thyroid Cancer, Childhood;Transitional Cell Cancer of the Renal Pelvis and Ureter; TrophoblasticTumor, Gestational; Unknown Primary Site, Cancer of, Childhood; UnusualCancers of Childhood; Ureter and Renal Pelvis, Transitional Cell Cancer;Urethral Cancer; Uterine Sarcoma; Vaginal Cancer; Visual Pathway andHypothalamic Glioma, Childhood; Vulvar Cancer; Waldenstrom's Macroglobulinemia; and Wilms' Tumor. Metastases of the aforementioned cancerscan also be treated or prevented in accordance with the methodsdescribed herein.

The methods described herein are useful in treating cancer in nervoussystem, e.g., brain tumor, e.g., glioma, e.g., glioblastoma multiforme(GBM), e.g., by inhibiting a neoactivity of a mutant enzyme, e.g., anenzyme in a metabolic pathway, e.g., a metabolic pathway leading tofatty acid biosynthesis, glycolysis, glutaminolysis, the pentosephosphate shunt, the nucleotide biosynthetic pathway, or the fatty acidbiosynthetic pathway, e.g., IDH1 or IDH2.

Gliomas, a type of brain tumors, can be classified as grade I to gradeIV on the basis of histopathological and clinical criteria establishedby the World Health Organization (WHO). WHO grade I gliomas are oftenconsidered benign. Gliomas of WHO grade II or III are invasive, progressto higher-grade lesions. WHO grade IV tumors (glioblastomas) are themost invasive form. Exemplary brain tumors include, e.g., astrocytictumor (e.g., pilocytic astrocytoma, subependymal giant-cell astrocytoma,diffuse astrocytoma, pleomorphic xanthoastrocytoma, anaplasticastrocytoma, astrocytoma, giant cell glioblastoma, glioblastoma,secondary glioblastoma, primary adult glioblastoma, and primarypediatric glioblastoma); oligodendroglial tumor (e.g.,oligodendroglioma, and anaplastic oligodendroglioma); oligoastrocytictumor (e.g., oligoastrocytoma, and anaplastic oligoastrocytoma);ependymoma (e.g., myxopapillary ependymoma, and anaplastic ependymoma);medulloblastoma; primitive neuroectodermal tumor, schwannoma,meningioma, meatypical meningioma, anaplastic meningioma; and pituitaryadenoma. Exemplary cancers are described in Acta Neuropathol (2008)116:597-602 and N Engl J Med. 2009 Feb. 19; 360(8):765-73, the contentsof which are each incorporated herein by reference.

In embodiments the disorder is glioblastoma.

In an embodiment the disorder is prostate cancer, e.g., stage T1 (e.g.,T1a, T1b and T1c), T2 (e.g., T2a, T2b and T2c), T3 (e.g., T3a and T3b)and T4, on the TNM staging system. In embodiments the prostate cancer isgrade G1, G2, G3 or G4 (where a higher number indicates greaterdifference from normal tissue). Types of prostate cancer include, e.g.,prostate adenocarcinoma, small cell carcinoma, squamous carcinoma,sarcomas, and transitional cell carcinoma.

Methods and compositions of the invention can be combined with art-knowntreatment. Art-known treatment for prostate cancer can include, e.g.,active surveillance, surgery (e.g., radical prostatectomy, transurethralresection of the prostate, orchiectomy, and cryosurgegry), radiationtherapy including brachytherapy (prostate brachytherapy) and externalbeam radiation therapy, High-Intensity Focused Ultrasound (HIFU),chemotherapy, cryosurgery, hormonal therapy (e.g., antiandrogens (e.g.,flutamide, bicalutamide, nilutamide and cyproterone acetate,ketoconazole, aminoglutethimide), GnRH antagonists (e.g., Abarelix)), ora combination thereof.

All references described herein are expressly incorporated herein byreference.

EXAMPLES Example 1 IDH1 Cloning, Mutagenesis, Expression andPurification

I. Wild Type IDH1 was Cloned into pET41a, Creating His8 Tag atC-Terminus.

The IDH1 gene coding region (cDNA) was purchased from Invitrogen inpENTR221 vector (www.invitrogen.com, Cat#B-068487_Ultimate_ORF). Oligonucleotides were designed to PCR out the coding region of IDH1 with NdeIat the 5′ end and XhoI at the 3′. (IDH1-f: TAATCATATGTCCAAAAAAATCAGT(SEQ ID NO:1), IDH1-r: TAATCTCGAGTGAAAGTTTGGCCTGAGCTAGTT (SEQ ID NO:2)).The PCR product is cloned into the NdeI/XhoI cleaved pET41a vector.NdeI/XhoI cleavage of the vector pET41a releases the GST portion of theplasmid, and creating a C-terminal His8 tag (SEQ ID NO:3) without theN-terminal GST fusion. The original stop codon of IDH1 is change toserine, so the junction sequence in final IDH1 protein is:Ser-Leu-Glu-His-His-His-His-His-His-His-His-Stop (SEQ ID NO:4).

The C-terminal His tag strategy instead of N-terminal His tag strategywas chosen, because C-terminal tag might not negatively impact IDH1protein folding or activity. See, e.g., Xu X et al, J Biol Chem. 2004Aug. 6; 279(32):33946-57.

The sequence for pET41a-IDH1 plasmid is confirmed by DNA sequencing.FIG. 1 shows detailed sequence verification of pET41a-IDH1 and alignmentagainst published IDH1 CDS below.

2. IDH1 Site Directed Mutagenesis to Create the IDHr132s and IDHr132hMutants.

Site directed mutagenesis was performed to convert R132 to S or H, DNAsequencing confirmed that G395 is mutated to A (creating Arg→Hismutation in the IDH1 protein), and C394 is mutated to A (creatingArg→Ser in the IDH1 protein). Detailed method for site directedmutagenesis is described in the user manual for QuikChange®MultiSite-Directed Mutagenesis Kit (Stratagene, cat#200531). FIG. 2shows DNA sequence verification of such mutations. Highlightednucleotides were successfully changed in the mutagenesis: G395→Amutation allows amino acid Arg132→His; C394→A mutation allows amino acidArg132→Ser.

3. IDH1 Protein Expression and Purification.

IDHwt, IDHR132S, and IDHR132H proteins were expressed in the E. colistrain Rosetta and purified according to the detailed procedure below.Active IDH1 proteins are in dimer form, and SEC column fraction/peakthat correspond to the dimer form were collected for enzymology analysisand cross comparison of catalytic activities of these proteins.

A. Cell Culturing:

Cells were grown in LB (20 μg/ml Kanamycin) at 37° C. with shaking untilOD600 reaches 0.6. The temperature was changed to 18° C. and protein wasinduced by adding IPTG to final concentration of 1 mM. Cells werecollected 12-16 hours after IPTG induction.

B. Buffer System:

Lysis buffer: 20 mM Tris, pH7.4, 0.1% Triton X-100, 500 mM NaCl, 1 mMPMSF, 5 mM β-mercaptoethanol, 10% glycerol.

Ni-Column Buffer A: 20 mM Tris, pH7.4, 500 mM NaCl, 5 mMβ-mercaptoethanol, 10% glycerol.

Ni-column Buffer B: 20 mM Tris, pH7.4, 500 mM NaCl, 5 mMβ-mercaptoethanol, 500 mM Imidazole, 10% glycerol

Gel filtration Buffer C: 200 mM NaCl, 50 mM Tris 7.5, 5 mMβ-mercaptoethanol, 2 mM MnSO₄, 10% glycerol.

C. Protein Purification Procedure

1. Cell pellet were resuspended in the lysis buffer (1 gram cell/5-10 mlbuffer).2. Cells were broken by passing the cell through Microfludizer with at apressure of 15,000 psi for 3 times.3. Soluble protein was collected from supernatant after centrifugationat 20,000 g (Beckman Avanti J-26XP) for 30 min at 4° C.4. 5-10 ml of Ni-column was equilibrated by Buffer A until the A280value reached baseline. The supernatant was loaded onto a 5-mlNi-Sepharose column (2 ml/min). The column was washed by 10-20 CV ofwashing buffer (90% buffer A+10% buffer B) until A280 reach the baseline(2 ml/min).5. The protein was eluted by liner gradient of 10-100% buffer B (20 CV)with the flow rate of 2 ml/min and the sample fractions were collectedas 2 ml/tube.6. The samples were analyzed on SDS-PAGE gel.7. The samples were collected and dialyzed against 200× Gel filtrationbuffer for 2 times (1 hour and >4 hours).8. The samples were concentrated to 10 ml.9. 200 ml of S-200 Gel-filtration column was equilibrated by buffer Cuntil the A280 value reached baseline. The samples were loaded onto Gelfiltration column (0.5 ml/min).10. The column was washed by 10 CV of buffer C, collect fractions as 2-4ml/tube.11. The samples were analyzed on SDS-PAGE gel and protein concentrationwas determined.

D. Protein Purification Results

The results for purification of wild type IDH1 are shown in FIGS. 3, 4,5A and 5B.

The results for purification of mutant IDH1R132S are shown in FIGS. 6,7, 8A and 8B.

The results for purification of wild type IDH1R132H are shown in FIGS.9, 10, 11A and 11B.

Example 2 Enzymology Analysis of IDH1 Wild Type and Mutants 1. Analysisof IDH1 Wild-Type and Mutants R132H and R132S in the OxidativeDecarboxylation of Isocitrate to α-Ketoglutarate (α-KG). A. Methods

To determine the catalytic efficiency of enzymes in the oxidativedecarboxylation of isocitrate to α-Ketoglutarate (α-KG) direction,reactions were performed to determine Vmax and Km for isocitrate. Inthese reactions, the substrate was varied while the cofactor was heldconstant at 500 uM. All reactions were performed in 150 mM NaCl, 20 mMTris-Cl, pH 7.5, 10% glycerol, and 0.03% (w/v) BSA). Reaction progresswas followed by spectroscopy at 340 nM monitoring the change inoxidation state of the cofactor. Sufficient enzyme was added to give alinear change in absorbance for 10 minutes.

B. ICDH1 R132H and ICDH1 R132S are Impaired for Conversion of Isocitrateto α-KG.

Michaelis-Menten plots for the relationship of isocitrate concentrationto reaction velocity are presented in FIGS. 12A-12C. Kinetic parametersare summarized in the Table 1. All data was fit to the Hill equation byleast-squares regression analysis.

TABLE 1 Relative Vmax Hill Catalytic Enzyme (umol/min/mg) Km (uM)Constant Vmax/Km Efficiency Wt 30.5 56.8 1.8 0.537    100% R132H 0.605171.7 0.6 0.0035   0.35% R132S 95 >1e6 0.479 <9.5e7 <.001%

Both mutant enzymes display a reduced Hill coefficient and an increasein Km for isocitrate, suggesting a loss of co-operativity in substratebinding and/or reduced affinity for substrate. R132H enzyme alsodisplays a reduced Vmax, suggestive of a lower kcat. R132S displays anincrease in Vmax, suggesting an increase in kcat, although this comes atthe expense of a 20,000 fold increase in Km so that the overall effecton catalytic efficiency is a great decrease as compared to the wild-typeenzyme. The relative catalytic efficiency, described as Vmax/Km, isdramatically lower for the mutants as compared to wild-type. The in vivoeffect of these mutations would be to decrease the flux conversion ofisocitrate to α-KG.

C. The ICDH1 R132H and R132S Mutants Display Reduced Product Inhibitionin the Oxidative Decarboxylation of Isocitrate to α-Ketoglutarate(α-KG).

A well-known regulatory mechanism for control of metabolic enzymes isfeedback inhibition, in which the product of the reaction acts as anegative regulator for the generating enzyme. To examine whether theR132S or R132H mutants maintain this regulatory mechanism, the Ki forα-KG in the oxidative decarboxylation of ioscitrate to α-ketoglutaratewas determined. Data is presented in FIGS. 13A-13C and summarized inTable 2. In all cases, α-KG acts as a competitive inhibitor of theisocitrate substrate. However, R132H and R132S display a 20-fold and13-fold increase in sensitivity to feedback inhibition as compared tothe wild-type enzyme.

TABLE 2 Enzyme Ki (uM) Wt 612.2 R132H 28.6 R132S 45.3

D. The Effect of MnCl₂ in Oxidative Decarboxylation of Isocitrate toα-Ketoglutarate (α-KG).

MnCl₂ can be substituted with MgCl₂ to examine if there is anydifference in oxidative decarboxylation of isocitrate to α-Ketoglutarate(α-KG).

E. The Effect of R132 Mutations on the Inhibitory Effect of Oxalomalateon IDH1

The purpose of this example is to examine the susceptibility ofIDH1R132S and IDH1R132H in oxidative decarboxylation of isocitrate toα-Ketoglutarate (α-KG) to the known IDH1 inhibitor oxalomalate.Experiments were performed to examine if R132 mutations circumvent theinhibition by oxalomalate.

Final concentrations: Tris 7.5 20 mM, NaCl 150 mM, MnCl₂ 2 mM, Glycerol10%, BSA 0.03%, NADP 0.5 mM, IDH1 wt 1.5 ug/ml, IDH1R132S 30 ug/ml,IDH1R132H 60 ug/ml, DL-isocitrate (5-650 uM). The results are summarizedin FIG. 17 and Table 3. The R132S mutation displays approximately atwo-fold increase in susceptibility to inhibition by oxalomalate, whilethe R132H mutation is essentially unaffected. In all three cases, thesame fully competitive mode of inhibition with regards to isocitrate wasobserved.

TABLE 3 Enzyme Oxalomalate Ki (uM) wt 955.4 R132S 510 R132H 950.8

F. Forward Reactions (Isocitrate to α-KG) of Mutant Enzyme do not go toCompletion.

Forward reactions containing ICDH1 R132S or ICDH1 R132H were assembledand reaction progress monitored by an increase in the OD340 of thereduced NADPH cofactor. It was observed (FIG. 23), that these reactionsproceed in the forward direction for a period of time and then reversedirection and oxidize the cofactor reduced in the early stages of thereaction, essentially to the starting concentration present at theinitiation of the experiment. Addition of further isocitratere-initiated the forward reaction for a period of time, but again didnot induce the reaction to proceed to completion. Rather, the systemreturned to initial concentrations of NADPH. This experiment suggestedthat the mutant enzymes were performing a reverse reaction other thanthe conversion of α-KG to isocitrate.

2. Analysis of IDH1 Wild-Type and Mutants R132H and R132S in theReduction of α-Ketoglutarate (α-KG). A. Methods

To determine the catalytic efficiency of enzymes in the reduction ofα-Ketoglutarate (α-KG), reactions were performed to determine Vmax andKm for α-KG. In these reactions, substrate was varied while the cofactorwas held constant at 500 uM. All reactions were performed in 50 mMpotassium phosphate buffer, pH 6.5, 10% glycerol, 0.03% (w/v) BSA, 5 mMMgCl₂, and 40 mM sodium hydrocarbonate. Reaction progress was followedby spectroscopy at 340 nM monitoring the change in oxidation state ofthe cofactor. Sufficient enzyme was added to give a linear change inabsorbance for 10 minutes.

B. The R132H and R132S Mutant Enzymes, but not the Wild-Type Enzyme,Support the Reduction of α-KG.

To test the ability of the mutant and wild-type enzymes to perform thereduction of α-KG, 40 ug/ml of enzyme was incubated under the conditionsfor the reduction of α-Ketoglutarate (α-KG) as described above. Resultsare presented in FIG. 14. The wild-type enzyme was unable to consumeNADPH, while R132S and R132H reduced α-KG and consumed NADPH.

C. The Reduction of α-KG by the R132H and R132S Mutants Occurs In Vitroat Physiologically Relevant Concentrations of α-KG.

To determine the kinetic parameters of the reduction of α-KG performedby the mutant enzymes, a substrate titration experiment was performed,as presented in FIGS. 15A-15B. R132H maintained the Hill-type substrateinteraction as seen in the oxidative decarboxylation of isocitrate, butdisplayed positive substrate co-operative binding. R132S showed aconversion to Michaelis-Menten kinetics with the addition ofuncompetitive substrate inhibition, as compared to wild-type enzyme inthe oxidative decarboxylation of isocitrate. The enzymatic parameters ofthe mutant enzyme are presented in Table 4. Since the wild-type enzymedid not consume measurable NADPH in the experiment described above, afull kinetic workup was not performed.

TABLE 4 Vmax Hill Enzyme (umol/min/mg) Km (mM) Constant Ki (mM) Vmax/KmR132H 1.3 0.965 1.8 1.35 R132S 2.7 0.181 0.479 24.6 14.92

The relative catalytic efficiency of reduction of α-KG is approximatelyten-fold higher in the R132S mutant than in the R132H mutant. Thebiological consequence is that the rate of metabolic flux should begreater in cells expressing R132S as compared to R132H.

D. Analysis of IDH1 Wild-Type and Mutants R132H and R132S in theReduction of Alpha-Ketoglutarate with NADH.

In order to evaluate the ability of the mutant enzymes to utilize NADHin the reduction of alpha-ketoglutarate, the following experiment wasconducted. Final concentrations: NaHCO3 40 mM, MgCl2 5 mM, Glycerol 10%,K2HPO4 50 mM, BSA 0.03%, NADH 0.5 mM, IDH1 wt 5 ug/ml, R132S 30 ug/ml,R132H 60 ug/ml, alpha-Ketoglutarate 5 mM.

The results are shown in FIG. 16 and Table 5. The R132S mutantdemonstrated the ability to utilize NADH while the wild type and R132Hshow no measurable consumption of NADH in the presence ofalpha-ketoglutarate.

TABLE 5 Consumption of NADH by R132S in the presence ofalpha-ketoglutarate R132S Mean SD Rate (ΔA/sec) 0.001117 0.0010880.001103 2.05E−05 Umol/min/mg 0.718328 0.699678 0.709003 0.013187

Summary

To understand how R132 mutations alter the enzymatic properties of IDH1,wild-type and R132H mutant IDH1 proteins were produced and purified fromE. coli. When NADP⁺-dependent oxidative decarboxylation of isocitratewas measured using purified wild-type or R132H mutant IDH1 protein, itwas confirmed that R132H mutation impairs the ability of IDH1 tocatalyze this reaction (Yan, H. et al. N Engl J Med 360, 765-73 (2009);Zhao, S. et al. Science 324, 261-5 (2009)), as evident by the loss inbinding affinity for both isocitrate and MgCl₂ along with a 1000-folddecrease in catalytic turnover (FIGS. 30A and 30C). In contrast, whenNADPH-dependent reduction of αKG was assessed using either wild-type orR132H mutant IDH1 protein, only R132H mutant could catalyze thisreaction at a measurable rate (FIGS. 30 and 30C). Part of this increasedrate of αKG reduction results from an increase in binding affinity forboth the cofactor NADPH and substrate αKG in the R132H mutant IDH1 (FIG.30C). Taken together, these data demonstrate that while the R132Hmutation leads to a loss of enzymatic function for oxidativedecarboxylation of isocitrate, this mutation also results in a gain ofenzyme function for the NADPH-dependent reduction of αKG.

2: Analysis of Mutant IDH1

The R132H Mutant does not Result in the Conversion of α-KG toIsocitrate.

Using standard experimental methods, an API2000 mass spectrometer wasconfigured for optimal detection of α-KG and isocitrate (Table 6). MRMtransitions were selected and tuned such that each analyte was monitoredby a unique transition. Then, an enzymatic reaction containing 1 mMα-KG, 1 mM NADPH, and ICDH1 R132H were assembled and run to completionas judged by the decrease to baseline of the optical absorbance at 340nM. A control reaction was performed in parallel from which the enzymewas omitted. Reactions were quenched 1:1 with methanol, extracted, andsubjected to analysis by LC-MS/MS.

FIG. 18A presents the control reaction indicating that aKG was notconsumed in the absence of enzyme, and no detectable isocitrate waspresent. FIG. 18B presents the reaction containing R132H enzyme, inwhich the α-KG has been consumed, but no isocitrate was detected. FIG.18C presents a second analysis of the reaction containing enzyme inwhich isocitrate has been spiked to a final concentration of 1 mM,demonstrating that had α-KG been converted to isocitrate at anyappreciable concentration greater than 0.01%, the configured analyticalsystem would have been capable of detecting its presence in the reactioncontaining enzyme. The conclusion from this experiment is that whileα-KG was consumed by R132H, isocitrate was not produced. This experimentindicates that one neoactivity of the R132H mutant is the reduction ofα-KG to a compound other than isocitrate.

TABLE 6 Instrument settings for MRM detection of compounds Compound Q1Q3 DP FP EP CEP CE CXP α-KG 144.975 100.6 −6 −220 −10 −16 −10 −22isocitrate 191.235 110.9 −11 −230 −4.5 −14 −16 −24 a-hydro- 147.085128.7 −11 −280 −10 −22 −12 −24 xyglutarate

The R132H Mutant Reduces α-KG to 2-Hydroxyglutaric Acid.

Using standard experimental methods, an API2000 mass spectrometer wasconfigured for optimal detection 2-hydroxyglutarate (Table 6 and FIG.19). The reaction products of the control and enzyme-containingreactions from above were investigated for the presence of2-hydroxyglutaric acid, FIG. 20. In the control reaction, no2-hydroxyglutaric acid was detected, while in reaction containing R132H,2-hydroxyglutaric acid was detected. This data confirms that oneneoactivity of the R132H mutant is the reduction of α-KG to2-hydroxyglutaric acid.

To determine whether R132H mutant protein directly produced 2HG fromαKG, the product of the mutant IDH1 reaction was examined using negativeion mode triple quadrupole electrospray LC-MS. These experimentsconfirmed that 2HG was the direct product of NADPH-dependent αKGreduction by the purified R132H mutant protein through comparison with aknown metabolite standards (FIG. 31A). Conversion of αKG to isocitratewas not observed.

One can determine the enantiomeric specificity of the reaction productthrough derivitization with DATAN (diacetyl-L-tartaric acid) andcomparing the retention time to that of known R and S standards. Thismethod is described in Struys et al. Clin Chem 50:1391-1395 (2004). Thestereo-specific production of either the R or S enantiomer ofalpha-hydroxyglutaric acid by ICDH1 R132H may modify the biologicalactivity of other enzymes present in the cell. The racemic productionmay also occur.

For example, one can measure the inhibitory effect ofalpha-hydroxyglutaric acid on the enzymatic activity of enzymes whichutilize α-KG as a substrate. In one embodiment, alpha-hydroxyglutaricacid may be a substrate- or product-analogue inhibitor of wild-typeICDH1. In another embodiment alpha-hydroxyglutaric acid may be asubstrate- or product-analogue inhibitor of HIF1 prolyl hydroxylase. Inthe former case, inhibition of wild type ICDH1 by the enzymatic productof R132H will reduce the circulating levels of aKG in the cell. In thelatter case, inhibition of HIF1 prolyl hydroxylase will result in thestabilization of HIF1 and an induction of the hypoxic response cohort ofcellular responses.

ICDH R132H Reduces aKG to the R-Enantiomer of 2-hydroxyglutarate.

There are two possible enantiomers of the ICDHR132H reductive reactionproduct, converting alpha-ketoglutarate to 2-hydroxyglutarate, with thechiral center being located at the alpha-carbon position. Exemplaryproducts are depicted below.

These are referred to by those with knowledge in the art as the R (orpro-R) and S (or pro-S) enantiomers, respectively. In order to determinewhich form or both is produced as a result of the ICDH1 neoactivitydescribed above, the relative amount of each chiral form in the reactionproduct was determined in the procedure described below.

Reduction of α-KG to 2-HG was performed by ICDHR132H in the presence ofNADPH as described above, and the reaction progress was monitored by achange in extinction coefficient of the nucleotide cofactor at 340 nM;once the reaction was judged to be complete, the reaction was extractedwith methanol and dried down completely in a stream of nitrogen gas. Inparallel, samples of chirally pure R-2-HG and a racemic mixture of R-and S-2-HG (produced by a purely chemical reduction of α-KG to 2-HG)were resuspended in ddH₂O, similarly extracted with methanol, and dried.

The reaction products or chiral standards were then resuspended in asolution of dichloromethane:acetic acid (4:1) containing 50 g/L DATANand heated to 75° C. for 30 minutes to promote the derivitization of2-HG in the scheme described below:

After cooling to room temperature, the derivitization reactions weredried to completion and resuspended in ddH₂O for analysis on an LC-MS/MSsystem. Analysis of reaction products and chiral standards was performedon an API2000 LC-MS/MS system using a 2×150 mM C18 column with anisocratic flow of 200 μl/min of 90:10 (ammonium formate, pH3.6:methanol) and monitoring the retention times of the 2-HG-DATANcomplex using XIC and the diagnostic MRM transition of 363/147 in thenegative ion mode.

It should be noted that retention times in the experiments describedbelow are approximate and accurate to within +/−1 minute; the highlyreproducible peak seen at 4 minutes is an artefact of a column switchingvalve whose presence has no result on the conclusions drawn from theexperiment.

Injection of the racemic mixture gave two peaks of equal area atretention times of 8 and 10 minutes (FIG. 24A), while injection of theR-2-HG standard resulted in a major peak of >95% area at 10 minutes anda minor peak <5% area at 8 minutes (FIG. 24B); indicating that theR-2-HG standard is approximately 95% R and 5% S. Thus, this methodallows us to separate the R and S-2-HG chiral forms and to determine therelative amounts of each in a given sample. Coinjection of the racemicmixture and the R-2-HG standard resulted in two peaks at 8 and 10minutes, with a larger peak at 10 minutes resulting from the addition ofsurplus pro-R-form (the standard) to a previously equal mixture of R-and S-2-HG (FIG. 24C). These experiments allow us to assign the 8 minutepeak to the S-2-HG form and the 10 minute peak to the R-2-HG form.

Injection of the derivatized neoactivity enzyme reaction product aloneyields a single peak at 10 minutes, suggesting that the neoactivityreaction product is chirally pure R-2-HG (FIG. 24D). Coinjection of theneoactivity reaction product with the R-2-HG standard results in a majorpeak of >95% area at 10 minutes (FIG. 24E) and a single minor peak of<5% area at 8 minutes (previously observed in injection of the R-2-HGstandard alone) confirming the chirality of the neoactivity product asR. Coinjection of a racemic mixture and the neoactivity reaction product(FIG. 24F) results in a 60% area peak at 10 minutes and a 40% area peakat 8 minutes; this deviation from the previously symmetrical peak areasobserved in the racemate sample being due to the excess presence ofR-2-HG form contributed by the addition of the neoactivity reactionproduct.

These experiments allow us to conclude that the ICDH1 neoactivity is ahighly specific chiral reduction of α-KG to R-2-HG.

Enzyme Properties of Other IDH1 Mutations

To determine whether the altered enzyme properties resulting from R132Hmutation were shared by other R132 mutations found in human gliomas,recombinant R132C, R132L and R132S mutant IDH1 proteins were generatedand the enzymatic properties assessed. Similar to R132H mutant protein,R132C, R132L, and R132S mutations all result in a gain-of-function forNADPH-dependent reduction of αKG (data not shown). Thus, in addition toimpaired oxidative decarboxylation of isocitrate, one common featureshared among the IDH1 mutations found in human gliomas is the ability tocatalyze direct NADPH-dependent reduction of αKG.

Identification of 2-HG Production in Glioblastoma Cell Lines Containingthe IDH-1 R132H Mutant Protein.

Generation of Genetic Engineered Glioblastoma Cell Lines ExpressingWildtype or Mutant IDH-1 Protein.

A carboxy-terminal Myc-DDK-tagged open reading frame (ORF) clone ofhuman isocitrate dehydrogenase 1 (IDH1; Ref. ID: NM_005896) cloned invector pCMV6 was obtained from commercial vendor Origen Inc. VectorpCMV6 contains both kanamycin and neomycin resistance cassettes forselection in both bacterial and mammalian cell systems. Standardmolecular biology mutagenesis techniques were utilized to alter the DNAsequence at base pair 364 of the ORF to introduce base pair change fromguanine to adenine resulting in a change in the amino acid code atposition 132 from argentine (wt) to histidine (mutant; or R132H).Specific DNA sequence alteration was confirmed by standard methods forDNA sequence analysis. Parental vector pCMV6 (no insert), pCMV6-wt IDH1or pCMV6-R132H were transfected into immortalized human glioblastomacell lines ATCC® CRL-2610 (LN-18) or HTB-14 (U-87) in standard growthmedium (DMEM; Dulbecco's modified Eagles Medium containing 10% fetalbovine serum). Approximately 24 hrs after transfection, the cellcultures were transitioned to DMEM containing G418 sodium salt atconcentrations of either 750 ug/ml (CRL-2610) or 500 ug/ml (HTB-14) toselect those cells in culture that expressed the integrated DNA cassetteexpressing both the neomycin selectable marker and the ORF for humanwild type or R132H. Pooled populations of G418 resistant cells weregenerated and expression of either wild type IDH1 or R132 IDH1 wasconfirmed by standard Western blot analysis of cell lysates usingcommercial antibodies recognizing either human IDH1 antigen or theengineered carboxy-terminal MYC-DDK expression tag. These stable clonalpools were then utilized for metaobolite preparation and analysis.

Procedure for Metabolite Preparation and Analysis.

Glioblastoma cell lines (CRL-2610 and HTB-14) expressing wildtype ormutant IDH-1 protein were grown using standard mammalian tissue culturetechniques on DMEM media containing 10% FCS, 25 mM glucose, 4 mMglutamine, and G418 antibiotic (CRL-2610 at 750 ug/mL; HTB-14 at 500ug/mL) to insure ongoing selection to preserve the transfected mutantexpression sequences. In preparation for metabolite extractionexperiments, cells were passaged into 10 cm round culture dishes at adensity of 1×10⁶ cells. Approximately 12 hours prior to metaboliteextraction, the culture media was changed (8 mL per plate) to DMEMcontaining 10% dialyzed FCS (10,000 mwco), 5 mM glucose, 4 mM glutamine,and G-418 antibiotic as before; the dialyzed FCS removes multiple smallmolecules form the culture media and enables cell culture-specificassessment of metabolite levels. The media was again changed 2 hoursprior to metabolite extraction. Metabolite extraction was accomplishedby quickly aspirating the media from the culture dishes in a sterilehood, immediately placing the dishes in a tray containing dry ice tocool them to −80° C., and as quickly as possible, adding 2.6 mL of 80%MeOH/20% water, pre-chilled to −80° C. in a dry-ice/acetone bath. Thesechilled, methanol extracted cells were then physically separated fromthe culture dish by scraping with a sterile polyethylene cell lifter(Corning #3008), brought into suspension and transferred to a 15 mLconical vial, then chilled to −20° C. An additional 1.0 mL of 80%MeOH/20% water was applied to the chilled culture dish and the celllifting procedure repeated, to give a final extraction volume of 3.6 mL.The extracts were centrifuged at 20,000×g for 30 minutes to sediment thecell debris, and 3.0 mL of the supernatants was transferred to ascrew-cap freezer vial and stored at −80° C. until ready for analysis.

In preparation for analysis, the extracts were removed from the freezerand dried on a nitrogen blower to remove methanol. The 100% aqueoussamples were analyzed by LCMS as follows. The extract (10 μL) wasinjected onto a reverse-phase HPLC column (Synergi 150 mm×2 mm,Phenomenex Inc.) and eluted using a linear gradient of LCMS-grademethanol (Buffer B) in Aq. 10 mM tributylamine, 15 mM Acetic acid(Buffer A), running from 3% Buffer B to 95% Buffer B over 45 minutes at200 μL/min. Eluted metabolite ions were detected using atriple-quadrapole mass spectrometer, tuned to detect in negative modewith multiple-reaction-monitoring mode transition set (MRM's) accordingto the molecular weights and fragmentation patterns for 38 known centralmetabolites, including 2-hydroxyglutarate (MRM parameters were optimizedby prior infusion of known compound standards). Data was processed usingAnalyst Software (Applied Biosystems, Inc.) and metabolite signalintensities were converted into absolute concentrations using signalbuild-up curves from injected mixtures of metabolite standards at knownconcentrations. Final metabolite concentrations were reported as mean ofat least three replicates, +/−standard deviation.

Results.

Analyses reveal significantly higher levels of 2-HG in cells thatexpress the IDH-1 R132H mutant protein. As shown in FIG. 26A, levels of2-HG in CRL-2610 cell lines expressing the IDH-1 R132H mutant proteinare approximately 28-fold higher than identical lines expressing thewild-type protein. Similarly, levels of 2-HG in HTB-14 cell linesexpressing the IDH-1 R132H mutant protein are approximately 38-foldhigher than identical lines expressing the wild-type protein, as shownin FIG. 26B.

Evaluation of 2-Hydroxyglutarate (2-HG) Production in Human GlioblastomaTumors Containing Mutations in Isocitrate Dehydrogenase 1 (IDH1) atAmino Acid 132.

Heterozygous somatic mutations at nucleotide position 395 (amino acidcodon 132) in the transcript encoding isocitrate dehydrogenase 1 (IDH1)can occur in brain tumors.

Tissue Source:

Human brain tumors were obtained during surgical resection, flash frozenin liquid nitrogen and stored at −80° C. Clinical classification of thetissue as gliomas was performed using standard clinical pathologycategorization and grading.

Genomic Sequence Analysis to Identify Brain Tumor Samples ContainingEither Wild Type Isocitrate Dehydrogenase (IDH1) or Mutations AlteringAmino Acid 132.

Genomic DNA was isolated from 50-100 mgs of brain tumor tissue usingstandard methods. A polymerase chain reaction (PCR) procedure was thenperformed on the isolated genomic DNA to amplify a 295 base pairfragment of the genomic DNA that contains both intron and 2^(nd) exonsequences of human IDH1 (FIG. 27). In FIG. 27, intron sequence is shownin lower case font; 2^(nd) exon IDH1 DNA sequence is shown in upper casefont; forward (5′) and reverse (3′) primer sequences are shown inunderlined font; guanine nucleotide mutated in a subset of human gliomatumors is shown in bold underlined font.

The amplified DNA fragment was then sequenced using standard protocolsand sequence alignments were performed to classify the sequences aseither wild type or mutant at the guanine nucleotide at base pair 170 ofthe amplified PCR fragment. Tumors were identified that containedgenomic DNA having either two copies of guanine (wild type) or a mixedor monoalellic combination of one IDH1 allele containing guanine and theother an adenine (mutant) sequence at base pair 170 of the amplifiedproduct (Table 15). The nucleotide change results in a change at aminoacid position 132 of human IDH1 protein from arginine (wild type) tohistidine (mutant) as has been previously reported.

TABLE 15 Sequence variance at base pair 170 of the amplified genomic DNAfrom human glioma samples. Sample Base IDH1 Amino ID 170 Acid 132Genotype 1102 G arginine wild type 1822 A histidine mutant  496 Garginine wild type 1874 A histidine mutant  816 A histidine mutant  534G arginine wild type AP-1 A histidine mutant AP-2 A histidine mutant

Procedure for Metabolite Preparation and Analysis.

Metabolite extraction was accomplished by adding a 10× volume (m/vratio) of −80 C methanol:water mix (80%:20%) to the brain tissue(approximately 100 mgs) followed by 30 s homogenization at 4 C. Thesechilled, methanol extracted homogenized tissues were then centrifuged at14,000 rpm for 30 minutes to sediment the cellular and tissue debris andthe cleared tissue supernatants were transferred to a screw-cap freezervial and stored at −80° C. For analysis, a 2× volume of tributylamine(10 mM) acetic acid (10 mM) pH 5.5 was added to the samples and analyzedby LCMS as follows. Sample extracts were filtered using a Millex-FG 0.20micron disk and 10 μL were injected onto a reverse-phase HPLC column(Synergi 150 mm×2 mm, Phenomenex Inc.) and eluted using a lineargradient LCMS-grade methanol (50%) with 10 mM tributylamine and 10 mMacetic acid) ramping to 80% methanol:10 mM tributylamine:10 mM aceticacid over 6 minutes at 200 μL/min Eluted metabolite ions were detectedusing a triple-quadrapole mass spectrometer, tuned to detect in negativemode with multiple-reaction-monitoring mode transition set (MRM's)according to the molecular weights and fragmentation patterns for 8known central metabolites, including 2-hydroxyglutarate (MRM parameterswere optimized by prior infusion of known compound standards). Data wasprocessed using Analyst Software (Applied Biosystems, Inc.) andmetabolite signal intensities were obtained by standard peak integrationmethods.

Results.

Analyses revealed dramatically higher levels of 2-HG in cells tumorsamples that express the IDH-1 R132H mutant protein. Data is summarizedin Table 16 and FIG. 28.

TABLE 16 Tumor Cells in Sam- Tumor ple Primary Specimen Foci Geno-Nucleotide 2HG □KG Malate Fumarate Succinate Isocitrate ID DiagnosisGrade (%) type change Codon (□mole/g) (□mole/g) (□mole/g) (□mole/g)(□mole/g) (□mole/g) 1 Glioblastoma, WHO n/a wild wild R132 0.18 0.1611.182 0.923 1.075 0.041 residual/recurrent grade type type IV 2Glioblastoma WHO n/a wild wild R132 0.16 0.079 1.708 1.186 3.156 0.100grade type type IV 3 Glioblastoma WHO n/a wild wild R132 0.13 0.0280.140 0.170 0.891 0.017 grade type type IV 4 Oligoastrocytoma WHO n/awild wild R132 0.21 0.016 0.553 1.061 1.731 0.089 grade type type II 5Glioblastoma WHO n/a mutant G364A R132H 16.97 0.085 1.091 0.807 1.3570.058 grade IV 6 Glioblastoma WHO n/a mutant G364A R132H 19.42 0.0230.462 0.590 1.966 0.073 grade IV 7 Glioblastoma WHO n/a mutant G364AR132H 31.56 0.068 0.758 0.503 2.019 0.093 grade IV 8 Oligodendroglioma,WHO 75 mutant G364A R132H 12.49 0.033 0.556 0.439 0.507 0.091 anaplasticgrade III 9 Oligodendroglioma, WHO 90 mutant G364A R132H 4.59 0.0291.377 1.060 1.077 0.574 anaplastic grade III 10 Oligoastrocytoma WHO n/amutant G364A R132H 6.80 0.038 0.403 0.503 1.561 0.065 grade II 11Glioblastoma WHO n/a wild wild R132 0.686 0.686 0.686 0.686 0.686 0.007grade type type IV 12 Glioblastoma WHO n/a mutant G364A R132H 18.79118.791 18.791 18.791 18.791 0.031 grade IV 13 Glioblastoma WHO n/amutant G364A R132H 4.59 0.029 1.377 1.060 1.077 0.043 grade IV 14Glioblastoma WHO n/a wild wild R132 0.199 0.046 0.180 0.170 0.221 0.014grade type type IV 15 Glioblastoma WHO n/a mutant C363G R132G 13.8270.030 0.905 0.599 1.335 0.046 grade IV 16 Glioblastoma WHO n/a mutantG364A R132H 28.364 0.068 0.535 0.488 2.105 0.054 grade IV 17Glioblastoma WHO n/a mutant C363A R132S 9.364 0.029 1.038 0.693 2.1510.121 grade IV 18 Glioblastoma WHO n/a wild wild R132 0.540 0.031 0.4680.608 1.490 0.102 grade type type IV 19 Glioma, malignant, WHO 80 mutantG364A R132H 19.000 0.050 0.654 0.391 2.197 0.171 astrocytoma grade IV 20Oligodendroglioma WHO 80 wild wild R132 0.045 0.037 1.576 0.998 1.4200.018 grade type type III 21 Glioma, malignant, WHO 95 wild wild R1320.064 0.034 0.711 0.710 2.105 0.165 astrocytoma grade type type IV 22Glioblastoma WHO 70 wild wild R132 0.171 0.041 2.066 1.323 0.027 0.072grade type type IV

To determine if 2HG production is characteristic of tumors harboringmutations in IDH1, metabolites were extracted from human malignantgliomas that were either wild-type or mutant for IDH1. It has beensuggested that αKG levels are decreased in cells transfected with mutantIDH1 (Zhao, S. et al. Science 324, 261-5 (2009)). The average αKG levelfrom 12 tumor samples harboring various R132 mutations was slightly lessthan the average αKG level observed in 10 tumors which are wild-type forIDH1. This difference in αKG was not statistically significant, and arange of αKG levels was observed in both wild-type and mutant tumors. Incontrast, increased 2HG levels were found in all tumors that containedan R132 IDH1 mutation. All R132 mutant IDH1 tumors examined had between5 and 35 μmol of 2HG per gram of tumor, while tumors with wild-type IDH1had over 100 fold less 2HG. This increase in 2HG in R132 mutant tumorswas statistically significant (p<0.0001). It was confirmed that (R)-2HGwas the isomer present in tumor samples (data not shown). Together thesedata establish that the novel enzymatic activity associated with R132mutations in IDH1 results in the production of 2HG in human brain tumorsthat harbor these mutations.

2HG is known to accumulate in the inherited metabolic disorder2-hydroxyglutaric aciduria. This disease is caused by deficiency in theenzyme 2-hydroxyglutarate dehydrogenase, which converts 2HG to αKG(Struys, E. A. et al. Am J Hum Genet 76, 358-60 (2005)). Patients with2-hydroxyglutarate dehydrogenase deficiencies accumulate 2HG in thebrain as assessed by MRI and CSF analysis, develop leukoencephalopathy,and have an increased risk of developing brain tumors (Aghili, M.,Zahedi, F. & Rafiee, J Neurooncol 91, 233-6 (2009); Kolker, S.,Mayatepek, E. & Hoffmann, G. F. Neuropediatrics 33, 225-31 (2002);Wajner, M., Latini, A., Wyse, A. T. & Dutra-Filho, C. S. J Inherit MetabDis 27, 427-48 (2004)). Furthermore, elevated brain levels of 2HG resultin increased ROS levels (Kolker, S. et al. Eur J Neurosci 16, 21-8(2002); Latini, A. et al. Eur J Neurosci 17, 2017-22 (2003)),potentially contributing to an increased risk of cancer. The ability of2HG to act as an NMDA receptor agonist may contribute to this effect(Kolker, S. et al. Eur J Neurosci 16, 21-8 (2002)). 2HG may also betoxic to cells by competitively inhibiting glutamate and/or αKGutilizing enzymes. These include transaminases which allow utilizationof glutamate nitrogen for amino and nucleic acid biosynthesis, andαKG-dependent prolyl hydroxylases such as those which regulate Hif1αlevels. Alterations in Hif1α have been reported to result from mutantIDH1 protein expression (Zhao, S. et al. Science 324, 261-5 (2009)).Regardless of mechanism, it appears likely that the gain-of-functionability of cells to produce 2HG as a result of R132 mutations in IDH1contributes to tumorigenesis. Patients with 2-hydroxyglutaratedehydrogenase deficiency have a high risk of CNS malignancy (Aghili, M.,Zahedi, F. & Rafiee, E. J Neurooncol 91, 233-6 (2009)). The ability ofmutant IDH1 to directly act on αKG may explain the prevalence of IDH1mutations in tumors from CNS tissue, which are unique in their highlevel of glutamate uptake and its ready conversion to αKG in the cytosol(Tsacopoulos, M. J Physiol Paris 96, 283-8 (2002)), thereby providinghigh levels of substrate for 2HG production. The apparent co-dominanceof the activity of mutant IDH1 with that of the wild-type enzyme isconsistent with the genetics of the disease, in which only a single copyof the gene is mutated. As discussed above, the wild-type IDH1 coulddirectly provide NADPH and αKG to the mutant enzyme. These data alsodemonstrate that mutation of R132 to histidine, serine, cysteine,glycine or leucine share a common ability to catalyze theNADPH-dependent conversion of αKG to 2HG. These findings help clarifywhy mutations at other amino acid residues of IDH1, including otherresidues essential for catalytic activity, are not found. Finally, thesefindings have clinical implications in that they suggest that 2HGproduction will identify patients with IDH1 mutant brain tumors. Thiswill be important for prognosis as patients with IDH1 mutations livelonger than patients with gliomas characterized by other mutations(Parsons, D. W. et al. Science 321, 1807-12 (2008)). In addition,patients with lower grade gliomas may benefit by the therapeuticinhibition of 2HG production. Inhibition of 2HG production by mutantIDH1 might slow or halt conversion of lower grade glioma into lethalsecondary glioblastoma, changing the course of the disease.

The Reaction Product of ICDH1 R132H Reduction of α-KG Inhibits theOxidative Decarboxylation of Isocitrate by Wild-Type ICDH1.

A reaction containing the wild-type ICDH1, NADP, and α-KG was assembled(under conditions as described above) to which was added in a titrationseries either (R)-2-hydroxyglutarate or the reaction product of theICDH1 R1321H mutant reduction of α-KG to 2-hydroxyglutarate. Thereaction product 2-HG was shown to inhibit the oxidative decarboxylationof isocitrate by the wild-type ICDH1, while the (R)-2-hydroxyglutaratedid not show any effect on the rate of the reaction. Since there areonly two possible chiral products of the ICDH1 R132H mutant reduction ofα-KG to 2-HG, and the (R)-2-HG did not show inhibition in this assay, itfollows that the product of the mutant reaction is the (S)-2-HG form.This experiment is presented in FIG. 25.

To determine the chirality of the 2HG produced, the products of theR132H reaction was derivatized with diacetyl-L-tartaric anhydride, whichallowed separating the (S) and (R) enantiomers of 2HG by simplereverse-phase LC and detecting the products by tandem mass spectrometry(Struys, E. A., Jansen, E. E., Verhoeven, N. M. & Jakobs, C. Clin Chem50, 1391-5 (2004)) (FIG. 31B). The peaks corresponding to the (S) and(R) isomers of 2HG were confirmed using racemic and R(−)-2HG standards.The reaction product from R132H co-eluted with R(−)-2HG peak,demonstrating that the R(−) stereoisomer is the product produced fromαKG by R132H mutant IDH1.

The observation that the reaction product of the mutant enzyme iscapable of inhibiting a metabolic reaction known to occur in cellssuggests that this reaction product might also inhibit other reactionswhich utilize α-KG, isocitrate, or citrate as substrates or produce themas products in vivo or in vitro.

Example 3 Metabolomics Analysis of IDH1 Wild Type and Mutants

Metabolomics research can provide mechanistic basis for why R132mutations confer survival advantage for GBM patients carrying suchmutations.

1. Metabolomics of GBM Tumor Cell Lines: Wild Type Vs R132 Mutants

Cell lines with R132 mutations can be identified and profiled.Experiments can be performed in proximal metabolite pool with a broadscope of metabolites.

2. Oxalomalate Treatment of GBM Cell Lines

Oxalomalate is a competitive inhibitor of IDH1. Change of NADPH(metabolomics) when IDH1 is inhibited by a small molecule can beexamined.

3. Metabolomics of Primary GBM Tumors: Wild Type Vs R132 Mutations

Primary tumors with R132 mutations can be identified. Experiments can beperformed in proximal metabolite pool with a broad scope of metabolites.

4. Detection of 2-hydroxyglutarate in Cells that Overexpress IDH1 132Mutants

Overexpression of an IDH1 132 mutant in cells may cause an elevatedlevel of 2-hydroxyglutarate and/or a reduced level ofalpha-ketoglutarate. One can perform a metabolomic experiment todemonstrate the consequence of this mutation on the cellular metabolitepool.

Example 4 Evaluation of IDH1 as a Cancer Target

shRNAmir inducible knockdown can be performed to examine the cellularphenotype and metabolomics profiles. HTS grade IDH1 enzymes areavailable. The IDH mutations described herein can be used for patientselection.

Example 5 siRNAs

IDH1

Exemplary siRNAs are presented in the following tables. Art-knownmethods can be used to select other siRNAs. siRNAs can be evaluated,e.g., by determining the ability of an siRNA to silence an IDH, e.g.,IDH1, e.g., in an in vitro system, e.g., in cultured cells, e.g., HeLacells or cultured glioma cells. siRNAs known in the art for silencingthe target can also be used, see, e.g., Silencing of cytosolic NADP+dependent isoccitrate dehydrogenase by small interfering RNA enhancesthe sensitivity of HeLa cells toward stauropine, Lee et al., 2009, FreeRadical Research, 43: 165-173.

The siRNAs in Table 7 (with the exception of entry 1356) were generatedusing the siRNA selection tool available on the worldwide web atjura.wi.mit.edu/bioc/siRNAext/. (Yuan et al. Nucl. Acids. Res. 200432:W130-W134.) Other selection tools can be used as well. Entry 1356 wasadapted from Silencing of cytosolic NADP+ dependent isoccitratedehydrogenase by small interfering RNA enhances the sensitivity of HeLacells toward stauropine, Lee et al., 2009, Free Radical Research, 43:165-173.

The siRNAs in Tables 7, 8, 9, 10, 11, 12, 13 and 14 represent candidatesspanning the IDH1 mRNA at nucleotide positions 628 and 629 according tothe sequence at GenBank Accession No. NM_005896.2 (SEQ ID NO:9, FIG.22).

The RNAs in the tables can be modified, e.g., as described herein.Modifications include chemical modifications to enhance properties,e.g., resistance to degradation, or the use of overhangs. For example,either one or both of the sense and antisense strands in the tables caninclude an additional dinucleotide at the 3′ end, e.g., TT, UU, dTdT.

TABLE 7 siRNAs targeting wildtype IDH1 Position on mRNA SEQ SEQ (FIG. IDID 21B) sense (5′ to 3′) NO: antisense (5′ to 3′) NO: 13GGUUUCUGCAGAGUCUACU 14 AGUAGACUCUGCAGAAACC 15 118 CUCUUCGCCAGCAUAUCAU 16AUGAUAUGCUGGCGAAGAG 17 140 GGCAGGCGAUAAACUACAU 18 AUGUAGUUUAUCGCCUGCC 19145 GCGAUAAACUACAUUCAGU 20 ACUGAAUGUAGUUUAUCGC 21 199GAAAUCUAUUCACUGUCAA 22 UUGACAGUGAAUAGAUUUC 23 257 GUUCUGUGGUAGAGAUGCA 24UGCAUCUCUACCACAGAAC 25 272 GCAAGGAGAUGAAAUGACA 26 UGUCAUUUCAUCUCCUUGC 27277 GGAGAUGAAAUGACACGAA 28 UUCGUGUCAUUUCAUCUCC 29 278GAGAUGAAAUGACACGAAU 30 AUUCGUGUCAUUUCAUCUC 31 280 GAUGAAAUGACACGAAUCA 32UGAUUCGUGUCAUUUCAUC 33 292 CGAAUCAUUUGGGAAUUGA 34 UCAAUUCCCAAAUGAUUCG 35302 GGGAAUUGAUUAAAGAGAA 36 UUCUCUUUAAUCAAUUCCC 37 332CCUACGUGGAAUUGGAUCU 38 AGAUCCAAUUCCACGUAGG 39 333 CUACGUGGAAUUGGAUCUA 40UAGAUCCAAUUCCACGUAG 41 345 GGAUCUACAUAGCUAUGAU 42 AUCAUAGCUAUGUAGAUCC 43356 GCUAUGAUUUAGGCAUAGA 44 UCUAUGCCUAAAUCAUAGC 45 408GGAUGCUGCAGAAGCUAUA 46 UAUAGCUUCUGCAGCAUCC 47 416 CAGAAGCUAUAAAGAAGCA 48UGCUUCUUUAUAGCUUCUG 49 418 GAAGCUAUAAAGAAGCAUA 50 UAUGCUUCUUUAUAGCUUC 51432 GCAUAAUGUUGGCGUCAAA 52 UUUGACGCCAACAUUAUGC 53 467CUGAUGAGAAGAGGGUUGA 54 UCAACCCUCUUCUCAUCAG 55 481 GUUGAGGAGUUCAAGUUGA 56UCAACUUGAACUCCUCAAC 57 487 GAGUUCAAGUUGAAACAAA 58 UUUGUUUCAACUUGAACUC 59495 GUUGAAACAAAUGUGGAAA 60 UUUCCACAUUUGUUUCAAC 61 502CAAAUGUGGAAAUCACCAA 62 UUGGUGAUUUCCACAUUUG 63 517 CCAAAUGGCACCAUACGAA 64UUCGUAUGGUGCCAUUUGG 65 528 CAUACGAAAUAUUCUGGGU 66 ACCCAGAAUAUUUCGUAUG 67560 GAGAAGCCAUUAUCUGCAA 68 UUGCAGAUAAUGGCUUCUC 69 614CUAUCAUCAUAGGUCGUCA 70 UGACGACCUAUGAUGAUAG 71 618 CAUCAUAGGUCGUCAUGCU 72AGCAUGACGACCUAUGAUG 73 621 CAUAGGUCGUCAUGCUUAU 74 AUAAGCAUGACGACCUAUG 75691 GAGAUAACCUACACACCAA 76 UUGGUGUGUAGGUUAUCUC 77 735CCUGGUACAUAACUUUGAA 78 UUCAAAGUUAUGUACCAGG 79 747 CUUUGAAGAAGGUGGUGGU 80ACCACCACCUUCUUCAAAG 81 775 GGGAUGUAUAAUCAAGAUA 82 UAUCUUGAUUAUACAUCCC 83811 GCACACAGUUCCUUCCAAA 84 UUUGGAAGGAACUGUGUGC 85 818GUUCCUUCCAAAUGGCUCU 86 AGAGCCAUUUGGAAGGAAC 87 844 GGUUGGCCUUUGUAUCUGA 88UCAGAUACAAAGGCCAACC 89 851 CUUUGUAUCUGAGCACCAA 90 UUGGUGCUCAGAUACAAAG 91882 GAAGAAAUAUGAUGGGCGU 92 ACGCCCAUCAUAUUUCUUC 93 942GUCCCAGUUUGAAGCUCAA 94 UUGAGCUUCAAACUGGGAC 95 968 GGUAUGAGCAUAGGCUCAU 96AUGAGCCUAUGCUCAUACC 97 998 GGCCCAAGCUAUGAAAUCA 98 UGAUUUCAUAGCUUGGGCC 991001 CCCAAGCUAUGAAAUCAGA 100 UCUGAUUUCAUAGCUUGGG 101 1127CAGAUGGCAAGACAGUAGA 102 UCUACUGUCUUGCCAUCUG 103 1133 GCAAGACAGUAGAAGCAGA104 UCUGCUUCUACUGUCUUGC 105 1184 GCAUGUACCAGAAAGGACA 106UGUCCUUUCUGGUACAUGC 107 1214 CCAAUCCCAUUGCUUCCAU 108 AUGGAAGCAAUGGGAUUGG109 1257 CCACAGAGCAAAGCUUGAU 110 AUCAAGCUUUGCUCUGUGG 111 1258CACAGAGCAAAGCUUGAUA 112 UAUCAAGCUUUGCUCUGUG 113 1262 GAGCAAAGCUUGAUAACAA114 UUGUUAUCAAGCUUUGCUC 115 1285 GAGCUUGCCUUCUUUGCAA 116UUGCAAAGAAGGCAAGCUC 117 1296 CUUUGCAAAUGCUUUGGAA 118 UUCCAAAGCAUUUGCAAAG119 1301 CAAAUGCUUUGGAAGAAGU 120 ACUUCUUCCAAAGCAUUUG 121 1307CUUUGGAAGAAGUCUCUAU 122 AUAGAGACUUCUUCCAAAG 123 1312 GAAGAAGUCUCUAUUGAGA124 UCUCAAUAGAGACUUCUUC 125 1315 GAAGUCUCUAUUGAGACAA 126UUGUCUCAAUAGAGACUUC 127 1356 GGACUUGGCUGCUUGCAUU 128 AAUGCAAGCAGCCAAGUCC129 1359 CUUGGCUGCUUGCAUUAAA 130 UUUAAUGCAAGCAGCCAAG 131 1371CAUUAAAGGUUUACCCAAU 132 AUUGGGUAAACCUUUAAUG 133 1385 CCAAUGUGCAACGUUCUGA134 UCAGAACGUUGCACAUUGG 135 1390 GUGCAACGUUCUGACUACU 136AGUAGUCAGAACGUUGCAC 137 1396 CGUUCUGACUACUUGAAUA 138 UAUUCAAGUAGUCAGAACG139 1415 CAUUUGAGUUCAUGGAUAA 140 UUAUCCAUGAACUCAAAUG 141 1422GUUCAUGGAUAAACUUGGA 142 UCCAAGUUUAUCCAUGAAC 143 1425 CAUGGAUAAACUUGGAGAA144 UUCUCCAAGUUUAUCCAUG 145 1455 CAAACUAGCUCAGGCCAAA 146UUUGGCCUGAGCUAGUUUG 147 1487 CCUGAGCUAAGAAGGAUAA 148 UUAUCCUUCUUAGCUCAGG149 1493 CUAAGAAGGAUAAUUGUCU 150 AGACAAUUAUCCUUCUUAG 151 1544CUGUGUUACACUCAAGGAU 152 AUCCUUGAGUGUAACACAG 153 1546 GUGUUACACUCAAGGAUAA154 UUAUCCUUGAGUGUAACAC 155 1552 CACUCAAGGAUAAAGGCAA 156UUGCCUUUAUCCUUGAGUG 157 1581 GUAAUUUGUUUAGAAGCCA 158 UGGCUUCUAAACAAAUUAC159 1646 GUUAUUGCCACCUUUGUGA 160 UCACAAAGGUGGCAAUAAC 161 1711CAGCCUAGGAAUUCGGUUA 162 UAACCGAAUUCCUAGGCUG 163 1713 GCCUAGGAAUUCGGUUAGU164 ACUAACCGAAUUCCUAGGC 165 1714 CCUAGGAAUUCGGUUAGUA 166UACUAACCGAAUUCCUAGG 167 1718 GGAAUUCGGUUAGUACUCA 168 UGAGUACUAACCGAAUUCC169 1719 GAAUUCGGUUAGUACUCAU 170 AUGAGUACUAACCGAAUUC 171 1725GGUUAGUACUCAUUUGUAU 172 AUACAAAUGAGUACUAACC 173 1730 GUACUCAUUUGUAUUCACU174 AGUGAAUACAAAUGAGUAC 175 1804 GGUAAAUGAUAGCCACAGU 176ACUGUGGCUAUCAUUUACC 177 1805 GUAAAUGAUAGCCACAGUA 178 UACUGUGGCUAUCAUUUAC179 1816 CCACAGUAUUGCUCCCUAA 180 UUAGGGAGCAAUACUGUGG 181 1892GGGAAGUUCUGGUGUCAUA 182 UAUGACACCAGAACUUCCC 183 1897 GUUCUGGUGUCAUAGAUAU184 AUAUCUAUGACACCAGAAC 185 1934 GCUGUGCAUUAAACUUGCA 186UGCAAGUUUAAUGCACAGC 187 1937 GUGCAUUAAACUUGCACAU 188 AUGUGCAAGUUUAAUGCAC189 1939 GCAUUAAACUUGCACAUGA 190 UCAUGUGCAAGUUUAAUGC 191 1953CAUGACUGGAACGAAGUAU 192 AUACUUCGUUCCAGUCAUG 193 1960 GGAACGAAGUAUGAGUGCA194 UGCACUCAUACUUCGUUCC 195 1961 GAACGAAGUAUGAGUGCAA 196UUGCACUCAUACUUCGUUC 197 1972 GAGUGCAACUCAAAUGUGU 198 ACACAUUUGAGUUGCACUC199 1976 GCAACUCAAAUGUGUUGAA 200 UUCAACACAUUUGAGUUGC 201 1982CAAAUGUGUUGAAGAUACU 202 AGUAUCUUCAACACAUUUG 203 1987 GUGUUGAAGAUACUGCAGU204 ACUGCAGUAUCUUCAACAC 205 1989 GUUGAAGAUACUGCAGUCA 206UGACUGCAGUAUCUUCAAC 207 2020 CCUUGCUGAAUGUUUCCAA 208 UUGGAAACAUUCAGCAAGG209 2021 CUUGCUGAAUGUUUCCAAU 210 AUUGGAAACAUUCAGCAAG 211 2024GCUGAAUGUUUCCAAUAGA 212 UCUAUUGGAAACAUUCAGC 213 2035 CCAAUAGACUAAAUACUGU214 ACAGUAUUUAGUCUAUUGG 215 2067 GAGUUUGGAAUCCGGAAUA 216UAUUCCGGAUUCCAAACUC 217 2073 GGAAUCCGGAAUAAAUACU 218 AGUAUUUAUUCCGGAUUCC219 2074 GAAUCCGGAAUAAAUACUA 220 UAGUAUUUAUUCCGGAUUC 221 2080GGAAUAAAUACUACCUGGA 222 UCCAGGUAGUAUUUAUUCC 223 2133 GGCCUGGCCUGAAUAUUAU224 AUAAUAUUCAGGCCAGGCC 225 2134 GCCUGAAUAUUAUACUACU 226AGUAGUAUAAUAUUCAGGC 227 2136 CUGGCCUGAAUAUUAUACU 228 AGUAUAAUAUUCAGGCCAG229 2166 CAUAUUUCAUCCAAGUGCA 230 UGCACUUGGAUGAAAUAUG 231 2180GUGCAAUAAUGUAAGCUGA 232 UCAGCUUACAUUAUUGCAC 233 2182 GCAAUAAUGUAAGCUGAAU234 AUUCAGCUUACAUUAUUGC 235 2272 CACUAUCUUAUCUUCUCCU 236AGGAGAAGAUAAGAUAGUG 237 2283 CUUCUCCUGAACUGUUGAU 238 AUCAACAGUUCAGGAGAAG239

TABLE 8 siRNAs targeting wildtype IDH1 Position on mRNA SEQ SEQ (FIG. IDID 21B) sense (5′ to 3′) NO: antisense (5′ to 3′) NO: 611AACCUAUCAUCAUAGGUCG 240 CGACCUAUGAUGAUAGGUU 241 612 ACCUAUCAUCAUAGGUCGU242 ACGACCUAUGAUGAUAGGU 243 613 CCUAUCAUCAUAGGUCGUC 244GACGACCUAUGAUGAUAGG 245 614 CUAUCAUCAUAGGUCGUCA 246 UGACGACCUAUGAUGAUAG247 615 UAUCAUCAUAGGUCGUCAU 248 AUGACGACCUAUGAUGAUA 249 616AUCAUCAUAGGUCGUCAUG 250 CAUGACGACCUAUGAUGAU 251 617 UCAUCAUAGGUCGUCAUGC252 GCAUGACGACCUAUGAUGA 253 618 CAUCAUAGGUCGUCAUGCU 254AGCAUGACGACCUAUGAUG 255 619 AUCAUAGGUCGUCAUGCUU 256 AAGCAUGACGACCUAUGAU257 620 UCAUAGGUCGUCAUGCUUA 258 UAAGCAUGACGACCUAUGA 259 621CAUAGGUCGUCAUGCUUAU 260 AUAAGCAUGACGACCUAUG 261 622 AUAGGUCGUCAUGCUUAUG262 CAUAAGCAUGACGACCUAU 263 623 UAGGUCGUCAUGCUUAUGG 264CCAUAAGCAUGACGACCUA 265 624 AGGUCGUCAUGCUUAUGGG 266 CCCAUAAGCAUGACGACCU267 625 GGUCGUCAUGCUUAUGGGG 268 CCCCAUAAGCAUGACGACC 269 626GUCGUCAUGCUUAUGGGGA 270 UCCCAUAAGCAUGACGACC 271 627 UCGUCAUGCUUAUGGGGAU272 AUCCCAUAAGCAUGACGAC 273

TABLE 9 siRNAs targeting G395A mutant IDH1 (SEQ ID NO: 5) (equivalent toG629A of SEQ ID NO: 9 (FIG. 21B)) Position SEQ on mRNA ID SEQ ID (FIG.21B) sense (5′ to 3′) NO: antisense (5′ to 3′) NO: 611AACCUAUCAUCAUAGGUCA 274 UGACCUAUGAUGAUAGGUU 275 612 ACCUAUCAUCAUAGGUCAU276 AUGACCUAUGAUGAUAGGU 277 613 CCUAUCAUCAUAGGUCAUC 278GAUGACCUAUGAUGAUAGG 279 614 CUAUCAUCAUAGGUCAUCA 280 UGAUGACCUAUGAUGAUAG281 615 UAUCAUCAUAGGUCAUCAU 282 AUGAUGACCUAUGAUGAUA 283 616AUCAUCAUAGGUCAUCAUG 284 CAUGAUGACCUAUGAUGAU 285 617 UCAUCAUAGGUCAUCAUGC286 GCAUGAUGACCUAUGAUGA 287 618 CAUCAUAGGUCAUCAUGCU 288AGCAUGAUGACCUAUGAUG 289 619 AUCAUAGGUCAUCAUGCUU 290 AAGCAUGAUGACCUAUGAU291 620 UCAUAGGUCAUCAUGCUUA 292 UAAGCAUGAUGACCUAUGA 293 621CAUAGGUCAUCAUGCUUAU 294 AUAAGCAUGAUGACCUAUG 295 622 AUAGGUCAUCAUGCUUAUG296 CAUAAGCAUGAUGACCUAU 297 623 UAGGUCAUCAUGCUUAUGG 298CCAUAAGCAUGAUGACCUA 299 624 AGGUCAUCAUGCUUAUGGG 300 CCCAUAAGCAUGAUGACCU301 625 GGUCAUCAUGCUUAUGGGG 302 CCCCAUAAGCAUGAUGACC 303 626GUCAUCAUGCUUAUGGGGA 304 UCCCCAUAAGCAUGAUGAC 305 627 UCAUCAUGCUUAUGGGGAU306 AUCCCCAUAAGCAUGAUGA 307

TABLE 10 siRNAs targeting C394A mutant IDH1 (SEQ ID NO: 5) (equivalentto C628A of SEQ ID NO: 9 (FIG. 21B)) (Arg132Ser (SEQ ID NO: 8)) PositionSEQ on mRNA ID SEQ ID (FIG. 21B) sense (5′ to 3′) NO: antisense (5′ to3′) NO: 611 AACCUAUCAUCAUAGGUAG 308 CUACCUAUGAUGAUAGGUU 309 612ACCUAUCAUCAUAGGUAGU 310 ACUACCUAUGAUGAUAGGU 311 613 CCUAUCAUCAUAGGUAGUC312 GACUACCUAUGAUGAUAGG 313 614 CUAUCAUCAUAGGUAGUCA 314UGACUACCUAUGAUGAUAG 315 615 UAUCAUCAUAGGUAGUCAU 316 AUGACUACCUAUGAUGAUA317 616 AUCAUCAUAGGUAGUCAUG 318 CAUGACUACCUAUGAUGAU 319 617UCAUCAUAGGUAGUCAUGC 320 GCAUGACUACCUAUGAUGA 321 618 CAUCAUAGGUAGUCAUGCU322 AGCAUGACUACCUAUGAUG 323 619 AUCAUAGGUAGUCAUGCUU 324AAGCAUGACUACCUAUGAU 325 620 UCAUAGGUAGUCAUGCUUA 326 UAAGCAUGACUACCUAUGA327 621 CAUAGGUAGUCAUGCUUAU 328 AUAAGCAUGACUACCUAUG 329 622AUAGGUAGUCAUGCUUAUG 330 CAUAAGCAUGACUACCUAU 331 623 UAGGUAGUCAUGCUUAUGG332 CCAUAAGCAUGACUACCUA 333 624 AGGUAGUCAUGCUUAUGGG 334CCCAUAAGCAUGACUACCU 335 625 GGUAGUCAUGCUUAUGGGG 336 CCCCAUAAGCAUGACUACC337 626 GUAGUCAUGCUUAUGGGGA 338 UCCCCAUAAGCAUGACUAC 339 627UAGUCAUGCUUAUGGGGAU 340 AUCCCCAUAAGCAUGACUA 341

TABLE 11 siRNAs targeting C394U mutant IDH1 (SEQ ID NO: 5) (equivalentto C628U of SEQ ID NO: 9 (FIG. 21B)) (Arg132Cys (SEQ ID NO: 8)) PositionSEQ on mRNA ID SEQ ID (FIG. 21B) sense (5′ to 3′) NO: antisense (5′ to3′) NO: 611 AACCUAUCAUCAUAGGUUG 342 CAACCUAUGAUGAUAGGUU 343 612ACCUAUCAUCAUAGGUUGU 344 ACAACCUAUGAUGAUAGGU 345 613 CCUAUCAUCAUAGGUUGUC346 GACAACCUAUGAUGAUAGG 347 614 CUAUCAUCAUAGGUUGUCA 348UGACAACCUAUGAUGAUAG 349 615 UAUCAUCAUAGGUUGUCAU 350 AUGACAACCUAUGAUGAUA351 616 AUCAUCAUAGGUUGUCAUG 352 CAUGACAACCUAUGAUGAU 353 617UCAUCAUAGGUUGUCAUGC 354 GCAUGACAACCUAUGAUGA 355 618 CAUCAUAGGUUGUCAUGCU356 AGCAUGACAACCUAUGAUG 357 619 AUCAUAGGUUGUCAUGCUU 358AAGCAUGACAACCUAUGAU 359 620 UCAUAGGUUGUCAUGCUUA 360 UAAGCAUGACAACCUAUGA361 621 CAUAGGUUGUCAUGCUUAU 362 AUAAGCAUGACAACCUAUG 363 622AUAGGUUGUCAUGCUUAUG 364 CAUAAGCAUGACAACCUAU 365 623 UAGGUUGUCAUGCUUAUGG366 CCAUAAGCAUGACAACCUA 367 624 AGGUUGUCAUGCUUAUGGG 368CCCAUAAGCAUGACAACCU 369 625 GGUUGUCAUGCUUAUGGGG 370 CCCCAUAAGCAUGACAACC371 626 GUUGUCAUGCUUAUGGGGA 372 UCCCCAUAAGCAUGACAAC 373 627UUGUCAUGCUUAUGGGGAU 374 AUCCCCAUAAGCAUGACAA 375

TABLE 12 siRNAs targeting C394G mutant IDH1 (SEQ ID NO: 5) (equivalentto C628G of SEQ ID NO: 9 (FIG. 21B)) (Arg132Gly (SEQ ID NO: 8)) Positionon mRNA (FIG. SEQ ID SEQ ID 21B) sense (5′ to 3′) NO: antisense (5′ to3′) NO: 611 AACCUAUCAUCAUAGGUGG 376 CCACCUAUGAUGAUAGGUU 377 612ACCUAUCAUCAUAGGUGGU 378 ACCACCUAUGAUGAUAGGU 379 613 CCUAUCAUCAUAGGUGGUC380 GACCACCUAUGAUGAUAGG 381 614 CUAUCAUCAUAGGUGGUCA 382UGACCACCUAUGAUGAUAG 383 615 UAUCAUCAUAGGUGGUCAU 384 AUGACCACCUAUGAUGAUA385 616 AUCAUCAUAGGUGGUCAUG 386 CAUGACCACCUAUGAUGAU 387 617UCAUCAUAGGUGGUCAUGC 388 GCAUGACCACCUAUGAUGA 389 618 CAUCAUAGGUGGUCAUGCU390 AGCAUGACCACCUAUGAUG 391 619 AUCAUAGGUGGUCAUGCUU 392AAGCAUGACCACCUAUGAU 393 620 UCAUAGGUGGUCAUGCUUA 394 UAAGCAUGACCACCUAUGA395 621 CAUAGGUGGUCAUGCUUAU 396 AUAAGCAUGACCACCUAUG 397 622AUAGGUGGUCAUGCUUAUG 398 CAUAAGCAUGACCACCUAU 399 623 UAGGUGGUCAUGCUUAUGG400 CCAUAAGCAUGACCACCUA 401 624 AGGUUGUCAUGCUUAUGGG 402CCCAUAAGCAUGACCACCU 403 625 GGUUGUCAUGCUUAUGGGG 404 CCCCAUAAGCAUGACCACC405 626 GUUGUCAUGCUUAUGGGGA 406 UCCCCAUAAGCAUGACCAC 407 627UUGUCAUGCUUAUGGGGAU 408 AUCCCCAUAAGCAUGACCA 409

TABLE 13 siRNAs targeting G395C mutant IDH1 (SEQ ID NO: 5) (equivalentto G629C of SEQ ID NO: 9 (FIG. 21B)) (Arg132Pro (SEQ ID NO: 8)) Positionon mRNA (FIG. SEQ ID SEQ ID 21B) sense (5′ to 3′) NO: antisense (5′ to3′) NO: 611 AACCUAUCAUCAUAGGUCG 410 CGACCUAUGAUGAUAGGUU 411 612ACCUAUCAUCAUAGGUCGU 412 ACGACCUAUGAUGAUAGGU 413 613 CCUAUCAUCAUAGGUCGUC414 GACGACCUAUGAUGAUAGG 415 614 CUAUCAUCAUAGGUCGUCA 416UGACGACCUAUGAUGAUAG 417 615 UAUCAUCAUAGGUCGUCAU 418 AUGACGACCUAUGAUGAUA419 616 AUCAUCAUAGGUCGUCAUG 420 CAUGACGACCUAUGAUGAU 421 617UCAUCAUAGGUCGUCAUGC 422 GCAUGACGACCUAUGAUGA 423 618 CAUCAUAGGUCGUCAUGCU424 AGCAUGACGACCUAUGAUG 425 619 AUCAUAGGUCGUCAUGCUU 426AAGCAUGACGACCUAUGAU 427 620 UCAUAGGUCGUCAUGCUUA 428 UAAGCAUGACGACCUAUGA429 621 CAUAGGUCGUCAUGCUUAU 430 AUAAGCAUGACGACCUAUG 431 622AUAGGUCGUCAUGCUUAUG 432 CAUAAGCAUGACGACCUAU 433 623 UAGGUCGUCAUGCUUAUGG434 CCAUAAGCAUGACGACCUA 435 624 AGGUCGUCAUGCUUAUGGG 436CCCAUAAGCAUGACGACCU 437 625 GGUCGUCAUGCUUAUGGGG 438 CCCCAUAAGCAUGACGACC439 626 GUCGUCAUGCUUAUGGGGA 440 UCCCCAUAAGCAUGACGAC 441 627UCGUCAUGCUUAUGGGGAU 442 AUCCCCAUAAGCAUGACGA 443

TABLE 14 siRNAs targeting G395U mutant IDH1 (SEQ ID NO: 5) (equivalentto G629U of SEQ ID NO: 9 (FIG. 21B)) (Arg132Leu (SEQ ID NO: 8)) Positionon mRNA (FIG. SEQ ID SEQ ID 21B) sense (5′ to 3′) NO: antisense (5′ to3′) NO: 611 AACCUAUCAUCAUAGGUCU 444 AGACCUAUGAUGAUAGGUU 445 612ACCUAUCAUCAUAGGUCUU 446 AAGACCUAUGAUGAUAGGU 447 613 CCUAUCAUCAUAGGUCUUC448 GAAGACCUAUGAUGAUAGG 449 614 CUAUCAUCAUAGGUCUUCA 450UGAAGACCUAUGAUGAUAG 451 615 UAUCAUCAUAGGUCUUCAU 452 AUGAAGACCUAUGAUGAUA453 616 AUCAUCAUAGGUCUUCAUG 454 CAUGAAGACCUAUGAUGAU 455 617UCAUCAUAGGUCUUCAUGC 456 GCAUGAAGACCUAUGAUGA 457 618 CAUCAUAGGUCUUCAUGCU458 AGCAUGAAGACCUAUGAUG 459 619 AUCAUAGGUCUUCAUGCUU 460AAGCAUGAAGACCUAUGAU 461 620 UCAUAGGUCUUCAUGCUUA 462 UAAGCAUGAAGACCUAUGA463 621 CAUAGGUCUUCAUGCUUAU 464 AUAAGCAUGAAGACCUAUG 465 622AUAGGUCUUCAUGCUUAUG 466 CAUAAGCAUGAAGACCUAU 467 623 UAGGUCUUCAUGCUUAUGG468 CCAUAAGCAUGAAGACCUA 469 624 AGGUCUUCAUGCUUAUGGG 470CCCAUAAGCAUGAAGACCU 471 625 GGUCUUCAUGCUUAUGGGG 472 CCCCAUAAGCAUGAAGACC473 626 GUCUUCAUGCUUAUGGGGA 474 UCCCCAUAAGCAUGAAGAC 475 627UCUUCAUGCUUAUGGGGAU 476 AUCCCCAUAAGCAUGAAGA 477

IDH2

Exemplary siRNAs are presented in the following tables. Art-knownmethods can be used to select other siRNAs. siRNAs can be evaluated,e.g., by determining the ability of an siRNA to silence an e.g., IDH2,e.g., in an in vitro system, e.g., in cultured cells, e.g., HeLa cellsor cultured glioma cells. e.g.,

The siRNAs in Table 15 were generated using the siRNA selection toolavailable on the worldwide web at jura.wi.mit.edu/bioc/siRNAext/. (Yuanet al. Nucl. Acids. Res. 2004 32:W130-W134.) Other selection tools canbe used as well. Entry 1356 was adapted from Silencing of cytosolicNADP+ dependent isoccitrate dehydrogenase by small interfering RNAenhances the sensitivity of HeLa cells toward stauropine, Lee et al.,2009, Free Radical Research, 43: 165-173.

The siRNAs in Tables 16-23 represent candidates spanning the IDH2 mRNAat nucleotide positions 600, 601, and 602 according to the mRNA sequencepresented at GenBank Accession No. NM_002168.2 (Record dated Aug. 16,2009; GI28178831) (SEQ ID NO12, FIG. 22B; equivalent to nucleotidepositions 514, 515, and 516 of the cDNA sequence represented by SEQ IDNO:11, FIG. FIG. 22A).

The RNAs in the tables can be modified, e.g., as described herein.Modifications include chemical modifications to enhance properties,e.g., resistance to degradation, or the use of overhangs. For example,either one or both of the sense and antisense strands in the tables caninclude an additional dinucleotide at the 3′ end, e.g., TT, UU, dTdT.

TABLE 15 siRNAs targeting wildtype IDH2 Position on mRNA (FIG. sense SEQID antisense SEQ ID 22B) (5′ to 3′) NO: (5′ to 3′) NO: 250GUGAUGAGAUGACCCGUAU 478 AUACGGGUCAUCUCAUCAC 479 252 GAUGAGAUGACCCGUAUUA480 UAAUACGGGUCAUCUCAUC 481 264 CGUAUUAUCUGGCAGUUCA 482UGAACUGCCAGAUAAUACG 483 274 GGCAGUUCAUCAAGGAGAA 484 UUCUCCUUGAUGAACUGCC485 451 GUGUGGAAGAGUUCAAGCU 486 AGCUUGAACUCUUCCACAC 487 453GUGGAAGAGUUCAAGCUGA 488 UCAGCUUGAACUCUUCCAC 489 456 GAAGAGUUCAAGCUGAAGA490 UCUUCAGCUUGAACUCUUC 491 795 CAGUAUGCCAUCCAGAAGA 492UCUUCUGGAUGGCAUACUG 493 822 CUGUACAUGAGCACCAAGA 494 UCUUGGUGCUCAUGUACAG495 832 GCACCAAGAACACCAUACU 496 AGUAUGGUGUUCUUGGUGC 497 844CCAUACUGAAAGCCUACGA 498 UCGUAGGCUUUCAGUAUGG 499 845 CAUACUGAAAGCCUACGAU500 AUCGUAGGCUUUCAGUAUG 501 868 GUUUCAAGGACAUCUUCCA 502UGGAAGAUGUCCUUGAAAC 503 913 CCGACUUCGACAAGAAUAA 504 UUAUUCUUGUCGAAGUCGG505 915 GACUUCGACAAGAAUAAGA 506 UCUUAUUCUUGUCGAAGUC 507 921GACAAGAAUAAGAUCUGGU 508 ACCAGAUCUUAUUCUUGUC 509 949 GGCUCAUUGAUGACAUGGU510 ACCAUGUCAUCAAUGAGCC 511 1009 GCAAGAACUAUGACGGAGA 512UCUCCGUCAUAGUUCUUGC 513 1010 CAAGAACUAUGACGGAGAU 514 AUCUCCGUCAUAGUUCUUG515 1024 GAGAUGUGCAGUCAGACAU 516 AUGUCUGACUGCACAUCUC 517 1096CUGAUGGGAAGACGAUUGA 518 UCAAUCGUCUUCCCAUCAG 519 1354 GCAAUGUGAAGCUGAACGA520 UCGUUCAGCUUCACAUUGC 521 1668 CUGUAAUUUAUAUUGCCCU 522AGGGCAAUAUAAAUUACAG 523 1694 CAUGGUGCCAUAUUUAGCU 524 AGCUAAAUAUGGCACCAUG525 1697 GGUGCCAUAUUUAGCUACU 526 AGUAGCUAAAUAUGGCACC 527 1698GUGCCAUAUUUAGCUACUA 528 UAGUAGCUAAAUAUGGCAC 529 1700 GCCAUAUUUAGCUACUAAA530 UUUAGUAGCUAAAUAUGGC 531

TABLE 16 siRNAs targeting wildtype IDH2 Position on mRNA (FIG. sense SEQID antisense SEQ ID 22B) (5′ to 3′) NO: (5′ to 3′) NO: 584GCCCAUCACCAUUGGCAGG 532 CCUGCCAAUGGUGAUGGGC 533 585 CCCAUCACCAUUGGCAGGC534 GCCUGCCAAUGGUGAUGGG 535 586 CCAUCACCAUUGGCAGGCA 536UGCCUGCCAAUGGUGAUGG 537 587 CAUCACCAUUGGCAGGCAC 538 GUGCCUGCCAAUGGUGAUG539 588 AUCACCAUUGGCAGGCACG 540 CGUGCCUGCCAAUGGUGAU 541 589UCACCAUUGGCAGGCACGC 542 GCGUGCCUGCCAAUGGUGA 543 590 CACCAUUGGCAGGCACGCC544 GGCGUGCCUGCCAAUGGUG 545 591 ACCAUUGGCAGGCACGCCC 546GGGCGUGCCUGCCAAUGGU 547 592 CCAUUGGCAGGCACGCCCA 548 UGGGCGUGCCUGCCAAUGG549 593 CAUUGGCAGGCACGCCCAU 550 AUGGGCGUGCCUGCCAAUG 551 594AUUGGCAGGCACGCCCAUG 552 CAUGGGCGUGCCUGCCAAU 553 595 UUGGCAGGCACGCCCAUGG554 CCAUGGGCGUGCCUGCCAA 555 596 UGGCAGGCACGCCCAUGGC 556GCCAUGGGCGUGCCUGCCA 557 597 GGCAGGCACGCCCAUGGCG 558 CGCCAUGGGCGUGCCUGCC559 598 GCAGGCACGCCCAUGGCGA 560 UCGCCAUGGGCGUGCCUGC 561 599CAGGCACGCCCAUGGCGAC 562 GUCGCCAUGGGCGUGCCUG 563 600 AGGCACGCCCAUGGCGACC564 GGUCGCCAUGGGCGUGCCU 565

TABLE 17 siRNAs targeting A514G mutant IDH2 (equivalent to A600G of SEQID NO: 12, (FIG. 22B) Position on mRNA (FIG. sense SEQ ID antisense SEQID 22B) (5′ to 3′) NO: (5′ to 3′) NO: 584 GCCCAUCACCAUUGGCGGG 566CCCGCCAAUGGUGAUGGGC 567 585 CCCAUCACCAUUGGCGGGC 568 GCCCGCCAAUGGUGAUGGG569 586 CCAUCACCAUUGGCGGGCA 570 UGCCCGCCAAUGGUGAUGG 571 587CAUCACCAUUGGCGGGCAC 572 GUGCCCGCCAAUGGUGAUG 573 588 AUCACCAUUGGCGGGCACG574 CGUGCCCGCCAAUGGUGAU 575 589 UCACCAUUGGCGGGCACGC 576GCGUGCCCGCCAAUGGUGA 577 590 CACCAUUGGCGGGCACGCC 578 GGCGUGCCCGCCAAUGGUG579 591 ACCAUUGGCGGGCACGCCC 580 GGGCGUGCCCGCCAAUGGU 581 592CCAUUGGCGGGCACGCCCA 582 UGGGCGUGCCCGCCAAUGG 583 593 CAUUGGCGGGCACGCCCAU584 AUGGGCGUGCCCGCCAAUG 585 594 AUUGGCGGGCACGCCCAUG 586CAUGGGCGUGCCCGCCAAU 587 595 UUGGCGGGCACGCCCAUGG 588 CCAUGGGCGUGCCCGCCAA589 596 UGGCGGGCACGCCCAUGGC 590 GCCAUGGGCGUGCCCGCCA 591 597GGCGGGCACGCCCAUGGCG 592 CGCCAUGGGCGUGCCCGCC 593 598 GCGGGCACGCCCAUGGCGA594 UCGCCAUGGGCGUGCCCGC 595 599 CGGGCACGCCCAUGGCGAC 596GUCGCCAUGGGCGUGCCCG 597 600 GGGCACGCCCAUGGCGACC 598 GGUCGCCAUGGGCGUGCCC599

TABLE 18 siRNAs targeting A514U mutant IDH2 (equivalent to A600U of SEQID NO: 12, (FIG. 22B) Position on mRNA (FIG. sense SEQ ID antisense SEQID 22B) (5′ to 3′) NO: (5′ to 3′) NO: 584 GCCCAUCACCAUUGGCUGG 600CCAGCCAAUGGUGAUGGGC 601 585 CCCAUCACCAUUGGCUGGC 602 GCCAGCCAAUGGUGAUGGG603 586 CCAUCACCAUUGGCUGGCA 604 UGCCAGCCAAUGGUGAUGG 605 587CAUCACCAUUGGCUGGCAC 606 GUGCCAGCCAAUGGUGAUG 607 588 AUCACCAUUGGCUGGCACG608 CGUGCCAGCCAAUGGUGAU 609 589 UCACCAUUGGCUGGCACGC 610GCGUGCCAGCCAAUGGUGA 611 590 CACCAUUGGCUGGCACGCC 612 GGCGUGCCAGCCAAUGGUG613 591 ACCAUUGGCUGGCACGCCC 614 GGGCGUGCCAGCCAAUGGU 615 592CCAUUGGCUGGCACGCCCA 616 UGGGCGUGCCAGCCAAUGG 617 593 CAUUGGCUGGCACGCCCAU618 AUGGGCGUGCCAGCCAAUG 619 594 AUUGGCUGGCACGCCCAUG 620CAUGGGCGUGCCAGCCAAU 621 595 UUGGCUGGCACGCCCAUGG 622 CCAUGGGCGUGCCAGCCAA623 596 UGGCUGGCACGCCCAUGGC 624 GCCAUGGGCGUGCCAGCCA 625 597GGCUGGCACGCCCAUGGCG 626 CGCCAUGGGCGUGCCAGCC 627 598 GCUGGCACGCCCAUGGCGA628 UCGCCAUGGGCGUGCCAGC 629 599 CUGGCACGCCCAUGGCGAC 630GUCGCCAUGGGCGUGCCAG 631 600 UGGCACGCCCAUGGCGACC 632 GGUCGCCAUGGGCGUGCCA633

TABLE 19 siRNAs targeting G515A mutant IDH2 (equivalent to G601A of SEQID NO: 12, (FIG. 22B) Position on mRNA (FIG. sense SEQ ID antisense SEQID 22B) (5′ to 3′) NO: (5′ to 3′) NO: 584 GCCCAUCACCAUUGGCAAG 634CUUGCCAAUGGUGAUGGGC 635 585 CCCAUCACCAUUGGCAAGC 636 GCUUGCCAAUGGUGAUGGG637 586 CCAUCACCAUUGGCAAGCA 638 UGCUUGCCAAUGGUGAUGG 639 587CAUCACCAUUGGCAAGCAC 640 GUGCUUGCCAAUGGUGAUG 641 588 AUCACCAUUGGCAAGCACG642 CGUGCUUGCCAAUGGUGAU 643 589 UCACCAUUGGCAAGCACGC 644GCGUGCUUGCCAAUGGUGA 645 590 CACCAUUGGCAAGCACGCC 646 GGCGUGCUUGCCAAUGGUG647 591 ACCAUUGGCAAGCACGCCC 648 GGGCGUGCUUGCCAAUGGU 649 592CCAUUGGCAAGCACGCCCA 650 UGGGCGUGCUUGCCAAUGG 651 593 CAUUGGCAAGCACGCCCAU652 AUGGGCGUGCUUGCCAAUG 653 594 AUUGGCAAGCACGCCCAUG 654CAUGGGCGUGCUUGCCAAU 655 595 UUGGCAAGCACGCCCAUGG 656 CCAUGGGCGUGCUUGCCAA657 596 UGGCAAGCACGCCCAUGGC 658 GCCAUGGGCGUGCUUGCCA 659 597GGCAAGCACGCCCAUGGCG 660 CGCCAUGGGCGUGCUUGCC 661 598 GCAAGCACGCCCAUGGCGA662 UCGCCAUGGGCGUGCUUGC 663 599 CAAGCACGCCCAUGGCGAC 664GUCGCCAUGGGCGUGCUUG 665 600 AAGCACGCCCAUGGCGACC 666 GGUCGCCAUGGGCGUGCUU667

TABLE 20 siRNAs targeting G515C mutant IDH2 (equivalent to G601C of SEQID NO: 12, (FIG. 22B) Position on mRNA (FIG. sense SEQ ID antisense SEQID 22B) (5′ to 3′) NO: (5′ to 3′) NO: 584 GCCCAUCACCAUUGGCACG 668CGUGCCAAUGGUGAUGGGC 669 585 CCCAUCACCAUUGGCACGC 670 GCGUGCCAAUGGUGAUGGG671 586 CCAUCACCAUUGGCACGCA 672 UGCGUGCCAAUGGUGAUGG 673 587CAUCACCAUUGGCACGCAC 674 GUGCGUGCCAAUGGUGAUG 675 588 AUCACCAUUGGCACGCACG676 CGUGCGUGCCAAUGGUGAU 677 589 UCACCAUUGGCACGCACGC 678GCGUGCGUGCCAAUGGUGA 679 590 CACCAUUGGCACGCACGCC 680 GGCGUGCGUGCCAAUGGUG681 591 ACCAUUGGCACGCACGCCC 682 GGGCGUGCGUGCCAAUGGU 683 592CCAUUGGCACGCACGCCCA 684 UGGGCGUGCGUGCCAAUGG 685 593 CAUUGGCACGCACGCCCAU686 AUGGGCGUGCGUGCCAAUG 687 594 AUUGGCACGCACGCCCAUG 688CAUGGGCGUGCGUGCCAAU 689 595 UUGGCACGCACGCCCAUGG 690 CCAUGGGCGUGCGUGCCAA691 596 UGGCACGCACGCCCAUGGC 692 GCCAUGGGCGUGCGUGCCA 693 597GGCACGCACGCCCAUGGCG 694 CGCCAUGGGCGUGCGUGCC 695 598 GCACGCACGCCCAUGGCGA696 UCGCCAUGGGCGUGCGUGC 697 599 CACGCACGCCCAUGGCGAC 698GUCGCCAUGGGCGUGCGUG 699 600 ACGCACGCCCAUGGCGACC 700 GGUCGCCAUGGGCGUGCGU701

TABLE 21 siRNAs targeting G515U mutant IDH2 (equivalent to G601U of SEQID NO: 12, (FIG. 22B) Position on mRNA (FIG. sense SEQ ID antisense SEQID 22B) (5′ to 3′) NO: (5′ to 3′) NO: 584 GCCCAUCACCAUUGGCAUG 702CAUGCCAAUGGUGAUGGGC 703 585 CCCAUCACCAUUGGCAUGC 704 GCAUGCCAAUGGUGAUGGG705 586 CCAUCACCAUUGGCAUGCA 706 UGCAUGCCAAUGGUGAUGG 707 587CAUCACCAUUGGCAUGCAC 708 GUGCAUGCCAAUGGUGAUG 709 588 AUCACCAUUGGCAUGCACG710 CGUGCAUGCCAAUGGUGAU 711 589 UCACCAUUGGCAUGCACGC 712GCGUGCAUGCCAAUGGUGA 713 590 CACCAUUGGCAUGCACGCC 714 GGCGUGCAUGCCAAUGGUG715 591 ACCAUUGGCAUGCACGCCC 716 GGGCGUGCAUGCCAAUGGU 717 592CCAUUGGCAUGCACGCCCA 718 UGGGCGUGCAUGCCAAUGG 719 593 CAUUGGCAUGCACGCCCAU720 AUGGGCGUGCAUGCCAAUG 721 594 AUUGGCAUGCACGCCCAUG 722CAUGGGCGUGCAUGCCAAU 723 595 UUGGCAUGCACGCCCAUGG 724 CCAUGGGCGUGCAUGCCAA725 596 UGGCAUGCACGCCCAUGGC 726 GCCAUGGGCGUGCAUGCCA 727 597GGCAUGCACGCCCAUGGCG 728 CGCCAUGGGCGUGCAUGCC 729 598 GCAUGCACGCCCAUGGCGA730 UCGCCAUGGGCGUGCAUGC 731 599 CAUGCACGCCCAUGGCGAC 732GUCGCCAUGGGCGUGCAUG 733 600 AUGCACGCCCAUGGCGACC 734 GGUCGCCAUGGGCGUGCAU735

TABLE 22 siRNAs targeting G516C mutant IDH2 (equivalent to G602C of SEQID NO: 12, (FIG. 22B) Position on mRNA (FIG. sense SEQ ID antisense SEQID 22B) (5′ to 3′) NO: (5′ to 3′) NO: 584 GCCCAUCACCAUUGGCAGC 736GCUGCCAAUGGUGAUGGGC 737 585 CCCAUCACCAUUGGCAGCC 738 GGCUGCCAAUGGUGAUGGG739 586 CCAUCACCAUUGGCAGCCA 740 UGGCUGCCAAUGGUGAUGG 741 587CAUCACCAUUGGCAGCCAC 742 GUGGCUGCCAAUGGUGAUG 743 588 AUCACCAUUGGCAGCCACG744 CGUGGCUGCCAAUGGUGAU 745 589 UCACCAUUGGCAGCCACGC 746GCGUGGCUGCCAAUGGUGA 747 590 CACCAUUGGCAGCCACGCC 748 GGCGUGGCUGCCAAUGGUG749 591 ACCAUUGGCAGCCACGCCC 750 GGGCGUGGCUGCCAAUGGU 751 592CCAUUGGCAGCCACGCCCA 752 UGGGCGUGGCUGCCAAUGG 753 593 CAUUGGCAGCCACGCCCAU754 AUGGGCGUGGCUGCCAAUG 755 594 AUUGGCAGCCACGCCCAUG 756CAUGGGCGUGGCUGCCAAU 757 595 UUGGCAGCCACGCCCAUGG 758 CCAUGGGCGUGGCUGCCAA759 596 UGGCAGCCACGCCCAUGGC 760 GCCAUGGGCGUGGCUGCCA 761 597GGCAGCCACGCCCAUGGCG 762 CGCCAUGGGCGUGGCUGCC 763 598 GCAGCCACGCCCAUGGCGA764 UCGCCAUGGGCGUGGCUGC 765 599 CAGCCACGCCCAUGGCGAC 766GUCGCCAUGGGCGUGGCUG 767 600 AGCCACGCCCAUGGCGACC 768 GGUCGCCAUGGGCGUGGCU769

TABLE 23 siRNAs targeting G516U mutant IDH2 (equivalent to G602U of SEQID NO: 12, (FIG. 22B) Position on mRNA (FIG. sense SEQ ID antisense SEQID 22B) (5′ to 3′) NO: (5′ to 3′) NO: 584 GCCCAUCACCAUUGGCAGU 770ACUGCCAAUGGUGAUGGGC 771 585 CCCAUCACCAUUGGCAGUC 772 GACUGCCAAUGGUGAUGGG773 586 CCAUCACCAUUGGCAGUCA 774 UGACUGCCAAUGGUGAUGG 775 587CAUCACCAUUGGCAGUCAC 776 GUGACUGCCAAUGGUGAUG 777 588 AUCACCAUUGGCAGUCACG778 CGUGACUGCCAAUGGUGAU 779 589 UCACCAUUGGCAGUCACGC 780GCGUGACUGCCAAUGGUGA 781 590 CACCAUUGGCAGUCACGCC 782 GGCGUGACUGCCAAUGGUG783 591 ACCAUUGGCAGUCACGCCC 784 GGGCGUGACUGCCAAUGGU 785 592CCAUUGGCAGUCACGCCCA 786 UGGGCGUGACUGCCAAUGG 787 593 CAUUGGCAGUCACGCCCAU788 AUGGGCGUGACUGCCAAUG 789 594 AUUGGCAGUCACGCCCAUG 790CAUGGGCGUGACUGCCAAU 791 595 UUGGCAGUCACGCCCAUGG 792 CCAUGGGCGUGACUGCCAA793 596 UGGCAGUCACGCCCAUGGC 794 GCCAUGGGCGUGACUGCCA 795 597GGCAGUCACGCCCAUGGCG 796 CGCCAUGGGCGUGACUGCC 797 598 GCAGUCACGCCCAUGGCGA798 UCGCCAUGGGCGUGACUGC 799 599 CAGUCACGCCCAUGGCGAC 800GUCGCCAUGGGCGUGACUG 801 600 AGUCACGCCCAUGGCGACC 802 GGUCGCCAUGGGCGUGACU803

Example 6 Structural Analysis of R132H Mutant IDH1

To define how R132 mutations alter the enzymatic properties of IDH1, thecrystal structure of R132H mutant IDH1 bound to αKG, NADPH, and Ca²⁺ wassolved at 2.1 Å resolution.

The overall quaternary structure of the homodimeric R132H mutant enzymeadopts the same closed catalytically competent conformation (shown as amonomer in FIG. 29A) that has been previously described for thewild-type enzyme (Xu, X. et al. J Biol Chem 279, 33946-57 (2004)). NADPHis positioned as expected for hydride transfer to αKG in an orientationthat would produce R(−)-2HG, consistent with our chiral determination ofthe 2HG product.

Two important features were noted by the change of R132 to histidine:the effect on catalytic conformation equilibrium and the reorganizationof the active-site. Locating atop a β-sheet in the relatively rigidsmall domain, R132 acts as a gate-keeper residue and appears toorchestrate the hinge movement between the open and closedconformations. The guanidinium moiety of R132 swings from the open tothe closed conformation with a distance of nearly 8 Å. Substitution ofhistidine for arginine is likely to change the equilibrium in favor ofthe closed conformation that forms the catalytic cleft for cofactor andsubstrate to bind efficiently, which partly explains the high-affinityfor NADPH exhibited by the R132H mutant enzyme. This feature may beadvantageous for the NADPH-dependent reduction of αKG to R(−)-2HG in anenvironment where NADPH concentrations are low. Secondly, closerexamination of the catalytic pocket of the mutant IDH1 structure incomparison to the wild-type enzyme showed not only the expected loss ofkey salt-bridge interactions between the guanidinium of R132 and the α/βcarboxylates of isocitrate, as well as changes in the network thatcoordinates the metal ion, but also an unexpected reorganization of theactive-site. Mutation to histidine resulted in a significant shift inposition of the highly conserved residues Y139 from the A subunit andK212′ from the B subunit (FIG. 29B), both of which are thought to becritical for catalysis of this enzyme family (Aktas, D. F. & Cook, P. F.Biochemistry 48, 3565-77 (2009)). In particular, the hydroxyl moiety ofY139 now occupies the space of the β-carboxylate of isocitrate. Inaddition, a significant repositioning of αKG compared to isocitratewhere the distal carboxylate of αKG now points upward to make newcontacts with N96 and S94 was observed. Overall, this single R132mutation results in formation of a distinct active site compared towild-type IDH1.

Example 7 Materials and Methods Summary

R132H, R132C, R132L and R132S mutations were introduced into human IDH1by standard molecular biology techniques. 293T and the humanglioblastoma cell lines U87MG and LN-18 were cultured in DMEM, 10% fetalbovine serum. Cells were transfected and selected using standardtechniques. Protein expression levels were determined by Western blotanalysis using IDHc antibody (Santa Cruz Biotechnology), IDH1 antibody(proteintech), MYC tag antibody (Cell Signaling Technology), and IDH2antibody (Abcam). Metabolites were extracted from cultured cells andfrom tissue samples according to close variants of a previously reportedmethod (Lu, W., Kimball, E. & Rabinowitz, J. D. J Am Soc Mass Spectrom17, 37-50 (2006)), using 80% aqueous methanol (−80° C.) and eithertissue scraping or homogenization to disrupt cells. Enzymatic activityin cell lysates was assessed by following a change in NADPH fluorescenceover time in the presence of isocitrate and NADP, or αKG and NADPH. Forenzyme assays using recombinant IDH1 enzyme, proteins were produced inE. coli and purified using Ni affinity chromatography followed bySephacryl S-200 size-exclusion chromatography. Enzymatic activity forrecombinant IDH1 protein was assessed by following a change in NADPH UVabsorbance at 340 nm using a stop-flow spectrophotometer in the presenceof isocitrate and NADP or αKG and NADPH. Chirality of 2HG was determinedas described previously (Struys, E. A., Jansen, E. E., Verhoeven, N. M.& Jakobs, C. Clin Chem 50, 1391-5 (2004)). For crystallography studies,purified recombinant IDH1 (R132H) at 10 mg/mL in 20 mM Tris pH 7.4, 100mM NaCl was pre-incubated for 60 min with 10 mM NADPH, 10 mM calciumchloride, and 75 mM αKG. Crystals were obtained at 20° C. by vapordiffusion equilibration using 3 μL drops mixed 2:1 (protein:precipitant)against a well-solution of 100 mM MES pH 6.5, 20% PEG 6000. Patienttumor samples were obtained after informed consent as part of a UCLAIRB-approved research protocol. Brain tumor samples were obtained aftersurgical resection, snap frozen in isopentane cooled by liquid nitrogenand stored at −80 C. The IDH1 mutation status of each sample wasdetermined using standard molecular biology techniques as describedpreviously (Yan, H. et al. N Engl J Med 360, 765-73 (2009)). Metaboliteswere extracted and analyzed by LC-MS/MS as described above. Full methodsare available in the supplementary material.

Supplementary Methods

Cloning, Expression, and Purification of ICDH1 wt and Mutants in E.coli.

The open reading frame (ORF) clone of human isocitrate dehydrogenase 1(cDNA) (IDH1; ref. ID NM_005896) was purchased from Invitrogen inpENTR221 (Carlsbad, Calif.) and Origene Inc. in pCMV6 (Rockville, Md.).To transfect cells with wild-type or mutant IDH1, standard molecularbiology mutagenesis techniques were utilized to alter the DNA sequenceat base pair 395 of the ORF in pCMV6 to introduce base pair change fromguanine to adenine, which resulted in a change in the amino acid code atposition 132 from arginine (wt) to histidine (mutant; or R132H), andconfirmed by standard DNA sequencing methods. For 293T celltransfection, wild-type and R132H mutant IDH1 were subcloned intopCMV-Sport6 with or without a carboxy-terminal Myc-DDK-tag. For stablecell line generation, constructs in pCMV6 were used. For expression inE. coli, the coding region was amplified from pENTR221 by PCR usingprimers designed to add NDEI and XHO1 restrictions sites at the 5′ and3′ ends respectively. The resultant fragment was cloned into vectorpET41a (EMD Biosciences, Madison, Wis.) to enable the E. coli expressionof C-terminus His8-tagged protein. Site directed mutagenesis wasperformed on the pET41a-ICHD1 plasmid using the QuikChange®MultiSite-Directed Mutagenesis Kit (Stratagene, La Jolla, Calif.) tochange G395 to A, resulting in the Arg to His mutation. R132C, R132L andR132S mutants were introduced into pET41a-ICHD1 in an analogous way.

Wild-type and mutant proteins were expressed in and purified from the E.coli Rosetta™ strain (Invitrogen, Carlsbad, Calif.) as follows. Cellswere grown in LB (20 μg/ml Kanamycin) at 37° C. with shaking until OD600reaches 0.6. The temperature was changed to 18° C. and proteinexpression was induced by adding IPTG to final concentration of 1 mM.After 12-16 hours of IPTG induction, cells were resuspended in LysisBuffer (20 mM Tris, pH7.4, 0.1% Triton X-100, 500 mM NaCl, 1 mM PMSF, 5mM β-mercaptoethanol, 10% glycerol) and disrupted by microfluidation.The 20,000 g supernatant was loaded on metal chelate affinity resin(MCAC) equilibrated with Nickel Column Buffer A (20 mM Tris, pH7.4, 500mM NaCl, 5 mM β-mercaptoethanol, 10% glycerol) and washed for 20 columnvolumes. Elution from the column was effected by a 20 column-volumelinear gradient of 10% to 100% Nickel Column Buffer B (20 mM Tris,pH7.4, 500 mM NaCl, 5 mM β-mercaptoethanol, 500 mM Imidazole, 10%glycerol) in Nickel Column Buffer A). Fractions containing the proteinof interest were identified by SDS-PAGE, pooled, and dialyzed twiceagainst a 200-volume excess of Gel Filtration Buffer (200 mM NaCl, 50 mMTris 7.5, 5 mM β-mercaptoethanol, 2 mM MnSO₄, 10% glycerol), thenconcentrated to 10 ml using Centricon (Millipore, Billerica, Mass.)centrifugal concentrators. Purification of active dimers was achieved byapplying the concentrated eluent from the MCAC column to a SephacrylS-200 (GE Life Sciences, Piscataway, N.J.) column equilibrated with GelFiltration Buffer and eluting the column with 20 column volumes of thesame buffer. Fractions corresponding to the retention time of thedimeric protein were identified by SDS-PAGE and pooled for storage at−80° C.

Cell Lines and Cell Culture.

293T cells were cultured in DMEM (Dulbecco's modified Eagles Medium)with 10% fetal bovine serum and were transfected using pCMV-6-basedIDH-1 constructs in six-well plates with Fugene 6 (Roche) orLipofectamine 2000 (Invitrogen) according to manufacturer'sinstructions. Parental vector pCMV6 (no insert), pCMV6-wt IDH1 orpCMV6-R132H were transfected into human glioblastoma cell lines (U87MG;LN-18 (ATCC, HTB-14 and CRL-2610; respectively) cultured in DMEM with10% fetal bovine serum. Approximately 24 hrs after transfection, thecell cultures were transitioned to medium containing G418 sodium salt atconcentrations of either 500 ug/ml (U87MG) or 750 ug/ml (LN-18) toselect stable transfectants. Pooled populations of G418 resistant cellswere generated and expression of either wild-type IDH1 or R132 IDH1 wasconfirmed by standard Western blot analysis.

Western Blot.

For transient transfection experiments in 293 cells, cells were lysed 72hours after transfection with standard RIPA buffer. Lysates wereseparated by SDS-PAGE, transferred to nitrocellulose and probed withgoat-anti-IDHc antibody (Santa Cruz Biotechnology sc49996) orrabbit-anti-MYC tag antibody (Cell Signaling Technology #2278) and thendetected with HRP-conjugated donkey anti-goat or HRP-conjugatedgoat-anti-rabbit antibody (Santa Cruz Biotechnology sc2004). IDH1antibody to confirm expression of both wild-type and R132H IDH1 wasobtained from Proteintech. The IDH2 mouse monoclonal antibody used wasobtained from Abcam.

Detection of Isocitrate, αKG, and 2HG in Purified Enzyme Reactions byLC-MS/MS.

Enzyme reactions performed as described in the text were run tocompletion as judged by measurement of the oxidation state of NADPH at340 nm. Reactions were extracted with eight volumes of methanol, andcentrifuged to remove precipitated protein. The supernatant was driedunder a stream of nitrogen and resuspended in H₂O. Analysis wasconducted on an API2000 LC-MS/MS (Applied Biosystems, Foster City,Calif.). Sample separation and analysis was performed on a 150×2 mm, 4uM Synergi Hydro-RP 80 A column, using a gradient of Buffer A (10 mMtributylamine, 15 mM acetic acid, 3% (v/v) methanol, in water) andBuffer B (methanol) using MRM transitions.

Cell Lysates Based Enzyme Assays.

293T cell lysates for measuring enzymatic activity were obtained 48hours after transfection with M-PER lysis buffer supplemented withprotease and phosphatase inhibitors. After lysates were sonicated andcentrifuged at 12,000 g, supernatants were collected and normalized fortotal protein concentration. To measure IDH oxidative activity, 3 μg oflysate protein was added to 200 μl of an assay solution containing 33 mMTris-acetate buffer (pH 7.4), 1.3 mM MgCl₂, 0.33 mM EDTA, 100 μM β-NADP,and varying concentrations of D-(+)-threo-isocitrate. Absorbance at 340nm, reflecting NADPH production, was measured every 20 seconds for 30min on a SpectraMax 190 spectrophotometer (Molecular Devices). Datapoints represent the mean activity of 3 replicates per lysate, averagedamong 5 time points centered at every 5 min. To measure IDH reductiveactivity, 3 μg of lysate protein was added to 200 μl of an assaysolution which contained 33 mM Tris-acetate (pH 7.4), 1.3 mM MgCl₂, 25μM β-NADPH, 40 mM NaHCO₃, and 0.6 mM αKG. The decrease in 340 nmabsorbance over time was measured to assess NADPH consumption, with 3replicates per lysate.

Recombinant IDH1 Enzyme Assays.

All reactions were performed in standard enzyme reaction buffer (150 mMNaCl, 20 mM Tris-Cl, pH 7.5, 10% glycerol, 5 mM MgCl₂ and 0.03% (w/v)bovine serum albumin). For determination of kinetic parameters,sufficient enzyme was added to give a linear reaction for 1 to 5seconds. Reaction progress was monitored by observation of the reductionstate of the cofactor at 340 nm in an SFM-400 stopped-flowspectrophotometer (BioLogic, Knoxville, Tenn.). Enzymatic constants weredetermined using curve fitting algorithms to standard kinetic modelswith the Sigmaplot software package (Systat Software, San Jose, Calif.).

Determination of Chirality of Reaction Products from Enzyme Reactionsand Tumors.

Enzyme reactions were run to completion and extracted with methanol asdescribed above, then derivatized with enantiomerically pure tartaricacid before resolution and analysis by LC-MS/MS. After being thoroughlydried, samples were resuspended in freshly prepared 50 mg/ml(2R,3R)-(+)-Tartaric acid in dichloromethane:acetic acid (4:1) andincubated for 30 minutes at 75° C. After cooling to room temperature,samples were briefly centrifuged at 14,000 g, dried under a stream ofnitrogen, and resuspended in H₂O. Analysis was conducted on an API200LC-MS/MS (Applied Biosystems, Foster City, Calif.), using an isocraticflow of 90:10 (2 mM ammonium formate, pH 3.6:MeOH) on a Luna C18(2)150×2 mm, 5 uM column. Tartaric-acid derivatized 2HG was detected usingthe 362.9/146.6 MRM transition and the following instrument settings: DP−1, FP −310, EP −4, CE-12, CXP-26. Analysis of the (R)-2HG standard, 2HGracemic mixture, and methanol-extracted tumor biomass (q.v.) wassimilarly performed.

Crystallography Conditions.

Crystals were obtained at 20° C. by vapor diffusion equilibration using3 μL drops mixed 2:1 (protein:precipitant) against a well-solution of100 mM MES pH 6.5, 20% PEG 6000.

Protein Characterization.

Approximately 90 mg of human cytosolic isocitrate dehydrogenase (HcIDH)was supplied to Xtal BioStructures by Agios. This protein was anengineered mutant form, R132S, with an 11-residue C-terminalaffinity-purification tag (sequence SLEHHHHHHHH). The calculatedmonomeric molecular weight was 48.0 kDa and the theoretical pI was 6.50.The protein, at about 6 mg/mL concentration, was stored in 1-mL aliquotsin 50 mM Tris-HCl (pH 7.4), 500 mM NaCl, 5 mM β-mercaptoethanol and 10%glycerol at −80° C. As shown in FIG. 32A, SDS-PAGE was performed to testprotein purity and an anti-histidine Western blot was done todemonstrate the protein was indeed his-tagged. A sample of the proteinwas injected into an FPLC size-exclusion column to evaluate the samplepurity and to determine the polymeric state in solution. FIG. 32B is achromatogram of this run showing a single peak running at an estimated87.6 kDa, suggesting IDH exists as a dimer at pH 7.4. Prior tocrystallization, the protein was exchanged into 20 mM Tris-HCl (pH 7.4)and 100 mM NaCl using Amicon centrifugal concentrators. At this time,the protein was also concentrated to approximately 15 mg/mL. At thisprotein concentration and ionic strength, the protein tended to form adetectable level of precipitate. After spinning out the precipitate, thesolution was stable at ˜10 mg/mL at 4° C.

Initial Attempts at Crystallization.

The strategy for obtaining diffraction-quality crystals was derived fromliterature conditions, specifically “Structures of Human CytosolicNADP-dependent Isocitrate Dehydrogenase Reveal a Novel Self-regulatoryMechanism of Activity,” Xu, et al. (2005) J. Biol. Chem. 279: 33946-56.In this study, two crystal forms of HcIDH wildtype protein wereproduced. One contained their “binary complex”, IDH-NADP, whichcrystallized from hanging drops in the tetragonal space group P4₃2₁2.The drops were formed from equal parts of protein solution (15 mg/mLIDH, 10 mM NADP) and precipitant consisting of 100 mM MES (pH 6.5) and12% PEG 20000. The other crystal form contained their “quaternarycomplex”, IDH-NADP/isocitrate/Ca^(2±), which crystallized in themonoclinic space group P2₁ using 100 mM MES (pH 5.9) and 20% PEG 6000 asthe precipitant. Here they had added 10 mM DL-isocitrate and 10 mMcalcium chloride to the protein solution. First attempts atcrystallizing the R132S mutant in this study centered around these tworeported conditions with little variation. The following lists thecomponents of the crystallization that could be varied; severaldifferent combinations of these components were tried in the screeningprocess.

In the protein solution:

HcIDH(R132S) always ˜10 mg/mL or ˜0.2 mMTris-HCl (pH 7.4) always 20 mMNaCl always 100 mMNADP⁺/NADPH absent or 5 mM NADP (did not try NADPH)DL-isocitic acid, trisodium salt absent or 5 mMcalcium chloride absent or 10 mMIn the precipitant: 100 mM MES (pH 6.5) and 12% PEG 20000

OR

-   -   100 mM MES (pH 6.0) and 20% PEG 6000        Drop size: always 3 μL        Drop ratios: 2:1, 1:1 or 1:2 (protein:precipitant)

Upon forming the hanging drops, a milky precipitate was always observed.On inspection after 2-4 days at 20° C. most drops showed denseprecipitation or phase separation. In some cases, the precipitatesubsided and it was from these types of drops small crystals had grown,for example, as shown in FIG. 33.

Crystal Optimization.

Once bonafide crystals were achieved, the next step was to optimize theconditions to obtain larger and more regularly-shaped crystals ofIDH-NADP/isocitrate/Ca²⁺ in a timely and consistent manner. The optimalscreen focused on varying the pH from 5.7 to 6.2, the MES concentrationfrom 50 to 200 mM and the PEG 6000 concentration from 20 to 25%. Also,bigger drops were set up (5-6 μl) and the drop ratios were again varied.These attempts failed to produce larger, diffraction-quality crystalsbut did reproduce the results reported above. Either a denseprecipitate, oily phase separation or small crystals were observed.

Using α-Ketoglutarate.

Concurrent to the optimization of the isocitrate crystals, other screenswere performed to obtain crystals of IDH(R132S) complexed withα-ketoglutarate instead. The protein solution was consistently 10 mg/mLIDH in 20 mM Tris-HCl (pH 7.4) and 100 mM NaCl. The following were addedin this order: 5 mM NADP, 5 mM α-ketoglutaric acid (free acid, pHbalanced with NaOH) and 10 mM calcium chloride. The protein was allowedto incubate with these compounds for at least an hour before the dropswere set up. The precipitant was either 100 mM MES (pH 6.5) and 12% PEG20000 or 100 mM MES (pH 6.5) and 20% PEG 6000. Again, precipitation orphase separation was primarily seen, but in some drops small crystalsdid form. At the edge of one of the drops, a single large crystalformed, pictured below. This was the single crystal used in thefollowing structure determination. FIG. 34 shows crystal obtained from aprotein solution contained 5 mM NADP, 5 mM α-ketoglutarate, 10 mM Ca2+.Precipitant contained 100 mM MES (pH 6.5) and 12% PEG 20000.

Cryo Conditions.

In order to ship the crystal to the X-ray source and protect it duringcryo-crystallography, a suitable cryo-protectant was needed. Glycerol isquite widely used and was the first choice. A cryo solution was made,basically as a mixture of the protein buffer and precipitant solutionplus glycerol: 20 mM Tris-HCl (pH 7.5), 100 mM NaCl, 5 mM NADP, 5 mMα-ketoglutaric acid, 10 mM calcium chloride, 100 mM MES (pH 6.5), 12%PEG 20000 and either 12.5% glycerol or 25% glycerol. The crystal wastransferred to the cryo solution in two steps. First, 5 μL of the 12.5%glycerol solution was added directly to the drop and incubated for 10minutes, watching for possible cracking of the crystal. The liquid wasremoved from the drop and 10 μL of the 25% glycerol solution was addedon top of the crystal. Again, this incubated for 10 minutes, harvestedinto a nylon loop and plunged into liquid nitrogen. The crystal wasstored submerged in a liquid nitrogen dewar for transport.

Data Collection and Processing.

The frozen crystal was mounted on a Rigaku RAXIS IV X-ray instrumentunder a stream of nitrogen gas at temperatures near −170° C. A 200°dataset was collected with the image plate detector using 1.54 Åwavelength radiation from a rotating copper anode home source, 1°oscillations and 10 minute exposures. The presence of 25% glycerol as acryoprotectant was sufficient for proper freezing, as no signs ofcrystal cracking (split spots or superimposed lattices) were observed. Adiffuse ring was observed at 3.6 Å resolution, most likely caused byicing. The X-ray diffraction pattern showed clear lattice planes andreasonable spot separation, although the spacing along one reciprocalaxis was rather small (b=275.3). The data was indexed to 2.7 Åresolution into space group P2₁2₁2 with HKL2000 (Otwinowski and Minor,1997). Three structures for HcIDH are known, designated the closed form(1T0L), the open form (1T09 subunit A) and semi-open form (1T09 subunitB). Molecular replacement was performed with the CCP4 program PHASER(Bailey, 1994) using only the protein atoms from these three forms. Onlythe closed form yielded a successful molecular replacement result with 6protein subunits in the asymmetric unit. The unit cell containsapproximately 53.8% solvent.

Model Refinement.

Using the CCP4 program REFMAC5, rigid-body refinement was performed tofit each of the 6 IDH subunits in the asymmetric unit. This was followedby rigid-body refinement of the three domains in each protein subunit.Restrained refinement utilizing non-crystallographic symmetry averagingof related pairs of subunits yielded an initial structure with R_(cryst)of 33% and R_(free) of 42%. Model building and real-space refinementwere performed using the graphics program COOT (Emsley and Cowtan,2004). A difference map was calculated and this showed strong electrondensity into which six individual copies of the NADP ligand and calciumion were manually fit with COOT. Density for the α-ketoglutaratestructure was less defined and was fit after the binding-site proteinresidues were fit using a 2F_(o)−F_(c) composite omit map. AutomatedRamachandran-plot optimization coupled with manual real-space densityfitting was applied to improve the overall geometry and fit. A finalround of restrained refinement with NCS yielded an R_(cryst) of 30.1%and R_(free) of 35.2%.

Unit cell a, Å b, Å c, Å α β γ volume, Å³ Z 116.14 275.30 96.28 90° 90°90° 3.08 × 10⁶ 24

Reflections in working 68,755/3,608 (5.0%) set/test set R_(cryst) 30.1%R_(free) 35.2%X-Ray Data and Refinement Statistics forIDH(R132S)-NADP/α-ketoglurate/Ca²⁺

Crystal parameters Space group P2₁2₁2 Unit cell dimensions a, b, c, Å116.139, 275.297, 96.283 α, β, γ, ° 90.0, 90.0, 90.0 Volume, Å³3,078,440 No. protein molecules in 6 asymmetric unit No. proteinmolecules in 24 unit cell, Z Data collection Beam line Date ofcollection Apr. 25, 2009 λ, Å 1.5418 Detector Rigaku Raxis IV Data set(phi), ° 200 Resolution, Å 25-2.7 (2.8-2.7) Unique reflections (N, F >0) 73,587 Completeness, % 85.4 (48.4) <I>/σI 9.88 (1.83) R-merge 0.109(0.33)  Redundancy 4.3 (1.8) Mosaicity 0.666 Wilson B factor 57.9Anisotropy B factor, Å² −1.96 Refinement Statistics Resolution limit, Å20.02-2.70 No. of reflections used 68,755/3608 for R-work^(a)/R-free^(b)Protein atoms 19788 Ligand atoms 348 No. of waters 357 Ions etc. 6Matthews coeff. Å³/ 2.68 Dalton Solvent, % 53.8 R-work^(a)/R-free^(b),(%) 30.1/35.2 Figure-of-merit^(c) 0.80 (0.74) Average B factors 31.0Coordinates error 0.484 (Luzzati plot), Å R.M.S. deviations Bondlengths, Å 0.026 Bond angles, ° 2.86

Completeness and R-merge are given for all data and for data in thehighest resolution shell. Highest shell values are in parentheses.

^(a)R factor=Σ_(hkl)|F_(o)−F_(c)|/Σ_(hkl)F_(o), where F_(o) and F_(c)are the observed and calculated structure factor amplitudes,respectively for all reflections hkl used in refinement.^(b)R-free is calculated for 5% of the data that were not used inrefinement.^(c)Figure of merit=√{square root over (x²+y²)}, where x=(Σ₀^(2π)P(α)cos α)/(Σ₀ ^(2π)P(α)), y=(Σ₀ ^(2π)P(α)sin α)/(Σ₀ ^(2π)P(α)),and the phase probability P(x)=exp(A cos α+B sin α+C cos(2α)+D sin(2α)),where A, B, C, and D are the Hendrickson-Lattman coefficients and cc isthe phase.

Stereochemistry of IDH(R132S)-NADP/α-ketoglurate/Ca²⁺ No. of amino % ofRamachandran plot statistics acids Residues Residues in most favoredregions [A, B, L] 1824 82.2 Residues in additional allowed regions [a,b, l, p] 341 15.4 Residues in generously allowed regions 38 1.7 [−a, −b,−l, −p] Residues in disallowed regions 17 0.8 Number of non-glycine andnon-proline residues 2220 100 Number of end-residues (excl. Gly and Pro)387 Number of glycine residues 198 Number of proline residues 72 Totalnumber of residues 2877 Overall <G>-factor^(d) score (>−1.0) −0.65

Generated by PROCHECK (Laskowski R A, MacArthur M W, Moss D S, ThorntonJ M (1993) J Appl Crystallogr 26:283-291.)

^(d)G-factors for main-chain and side-chain dihedral angles, andmain-chain covalent forces (bond lengths and bond angles). Values shouldbe ideally −0.5 or above −1.0.

Radiation wavelength, Å 1.54 Resolution, Å (outer shell) 20-2.70(2.80-2.70) Unique reflections 73,587 Completeness (outer shell) 85.4%(48.4%) Redundancy (outer shell) 4.3 (1.8) R_(merge) (outer shell) 10.9%(33%) <I>/<σ(I)> (outer shell) 9.88 (1.83)

Clinical Specimens, Metabolite Extraction and Analysis.

Human brain tumors were obtained during surgical resection, snap frozenin isopentane cooled by liquid nitrogen and stored at −80 C. Clinicalclassification of the tissue was performed using standard clinicalpathology categorization and grading as established by the WHO. Genomicsequence analysis was deployed to identify brain tumor samplescontaining either wild-type isocitrate dehydrogenase (IDH1) or mutationsaltering amino acid 132. Genomic DNA was isolated from 50-100 mgs ofbrain tumor tissue using standard methods. A polymerase chain reactionon the isolated genomic DNA was used to amplify a 295 base pair fragmentof the genomic DNA that contains both the intron and 2^(nd) exonsequences of human IDH1 and mutation status assessed by standardmolecular biology techniques.

Metabolite extraction was accomplished by adding a 10× volume (m/vratio) of −80° C. methanol:water mix (80%:20%) to the brain tissue(approximately 100 mgs) followed by 30 s homogenization at 4 C. Thesechilled, methanol extracted homogenized tissues were then centrifuged at14,000 rpm for 30 minutes to sediment the cellular and tissue debris andthe cleared tissue supernatants were transferred to a screw-cap freezervial and stored at −80° C. For analysis, a 2× volume of tributylamine(10 mM) acetic acid (10 mM) pH 5.5 was added to the samples and analyzedby LCMS as follows. Sample extracts were filtered using a Millex-FG 0.20micron disk and 10 μL were injected onto a reverse-phase HPLC column(Synergi 150 mm×2 mm, Phenomenex Inc.) and eluted using a lineargradient LCMS-grade methanol (50%) with 10 mM tributylamine and 10 mMacetic acid) ramping to 80% methanol:10 mM tributylamine:10 mM aceticacid over 6 minutes at 200 μL/min Eluted metabolite ions were detectedusing a triple-quadrupole mass spectrometer, tuned to detect in negativemode with multiple-reaction-monitoring mode transition set (MRM's)according to the molecular weights and fragmentation patterns for 8known central metabolites, including 2-hydroxyglutarate as describedabove. Data was processed using Analyst Software (Applied Biosystems,Inc.) and metabolite signal intensities were obtained by standard peakintegration methods.

Example 9 Compounds that Inhibit IDH1 R132H

Assays were conducted in a volume of 76 ul assay buffer (150 mM NaCl, 10mM MgCl2, 20 mM Tris pH 7.5, 0.03% bovine serum albumin) as follows in astandard 384-well plate: To 25 ul of substrate mix (8 uM NADPH, 2 mMaKG), 1 ul of test compound was added in DMSO. The plate was centrifugedbriefly, and then 25 ul of enzyme mix was added (0.2 ug/ml ICDH1 R132H)followed by a brief centrifugation and shake at 100 RPM. The reactionwas incubated for 50 minutes at room temperature, then 25 ul ofdetection mix (30 uM resazurin, 36 ug/ml) was added and the mixturefurther incubated for 5 minutes at room temperature. The conversion ofresazurin to resorufin was detected by fluorescent spectroscopy at Ex544Em590 c/o 590.

Table 24a shows the wild type vs mutant selectivity profile of 5examples of IDH1R132H inhibitors. The IDH1 wt assay was performed at1×Km of NADPH as opposed to IDHR132H at 10× or 100×Km of NADPH. Thesecond example showed no inhibition, even at 100 uM. Also, the firstexample has IC50=5.74 uM but is shifted significantly when assayed at100×Km, indicating direct NADPH-competitive inhibitor. The selectivitybetween wild type vs mutant could be >20-fold.

TABLE 24a ICDH IC50 ICDH (uM) @ IC50 4 uM (uM) @ IC50 IDH1wt LDHa LDHb(10x Km) 40 uM Ratio IC50 @ 1x STRUCTURE IC50 IC50 NADPH NADPH (40/4) Km(uM)

25.43 64.07  5.74 >100 17.42 16.22

 5.92 17.40 12.26 41.40  3.38 NO inhibition

 8.61 >100 12.79 14.70  1.15 19.23

33.75 >100 14.98 19.17  1.28 46.83

12.76 >100 23.80 33.16  1.39 69.33

Additional exemplary compounds that inhibit IDH1R132H are provided belowin Table 24b.

Compound No.

 1

 2

 3

 4

 5

 6

 7

 8

 9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

46

47

48

49

50

51

52

53

54

55

56

57

58

59

60

61

62

63

64

65

66

67

68

69

70

71

72

73

74

75

76

77

78

79

80

81

82

83

84

85

86

87

88

89

90

91

92

Example 10. The Mutant Enzyme IDH2-R172K has Elevated NADPH ReductiveCatalysis Activity as Compared to Wildtype IDH2 Enzyme

NADPH reduction activity was measured for the enzymes IDH2-R172K,IDH2-wildtype, IDH1-R132H and IDH1-wildtype. The final reactantconcentrations for each reaction were as follows: 20 mM Tris 7.5, 150 mMNaCl, 2 mM MnCl₂, 10% glycerol, 0.03% BSA, enzyme (1-120 μg/mL), 1 mMNADPH, and 5 mM aKG (alpha ketoglutarate). The resulting specificactivities (μmol/min/mg) are presented in the graph in FIG. 35. Theresults indicate that the mutant IDH2 has elevated reductive activity ascompared to wildtype IDH2, even though both the mutant and wildtype IDH2enzymes were able to make 2HG (2-hydroxyglutarate) at saturating levelsof reactants aKG and NADPH.

Example 11: 2-HG Accumulates in AML with IDH1/2 Mutations Patients andClinical Data

Peripheral blood and bone marrow were collected from AML patients at thetime of diagnosis and at relapse, following REB approved informedconsent. The cells were separated by ficol hypaque centrifugation, andstored at −150° C. in 10% DMSO, 40% FCS and 50% alpha-MEM medium.Patient sera were stored at −80° C. Cytogenetics and molecular testingwere performed in the diagnostic laboratory of the University HealthNetwork (Toronto, Canada). A subgroup of patients (n=132) was givenconsistent initial treatment using a standard induction andconsolidation chemotherapy regimen consisting of daunorubicin andcytarabine.

IDH1 and IDH2 Genotyping

DNA was extracted from leukemic cells and cell lines using the QiagenPuregene kit (Valencia Calif.). For a subset of samples (n=96), RNA wasextracted from leukemic cells using a Qiagen RNeasy kit, and reversetranscribed into cDNA for IDH1 and IDH2 genotyping. IDH1 and IDH2genotype was determined at the Analytical Genetics Technology Centre atthe University Health Network (Toronto, Canada) using a SequenomMassARRAY™ platform (Sequenom, San Diego, Calif.). Positive results wereconfirmed by direct sequencing on an ABI PRISM 3130XL genetic analyzer(Applied Biosystems, Foster City, Calif.).

Cell Lines

AML cell lines (OCI/AML-1, OCI/AML-2, OCI/AML-3, OCI/AML-4, OCI/AML-5,HL-60, MV-4-11, THP-1, K562, and KG1A) and 5637 cells were obtained fromthe laboratory of Mark Minden (Ontario Cancer Institute, Toronto,Canada). Primary AML cells were cultured in alpha-MEM media supplementedwith 20% fetal bovine serum, and 10% 5637 cell conditioned media aspreviously described¹³. Growth curves were generated by counting viablecells as assessed by trypan blue exclusion on a Vi-CELL automated cellcounter (Beckman Coulter, Fullarton, Calif.).

Expression/Purification of IDH1 and IDH2 Proteins

The human IDH1 cDNA (ref. ID NM_005896) and IDH2 cDNA (ref. IDNM_002168) were purchased from OriGene Technologies (Rockville, Md.).For expression in E. coli, the coding region was amplified by PCR usingprimers designed to add NDEI and XHO1 restrictions sites at the 5′ and3′ ends respectively. The resultant fragments for IDH1 (full length) andIDH2 (residues 40-452) were cloned into vector pET41a (EMD Biosciences,Madison, Wis.) to enable the E. coli expression of C-terminalHis8-tagged protein. Site directed mutagenesis was performed on thepET41a-IDH1 and pET41a-IDH2 plasmid using the QuikChange® LightningSite-Directed Mutagenesis Kit (Stratagene, La Jolla, Calif.) to changeC394 to T in the IDH1 cDNA, resulting in the R132C mutation, and tochange G515 to A in the IDH2 cDNA, resulting in the R172K mutation.Wild-type and mutant IDH1 proteins were expressed in and purified fromthe E. coli Rosetta™ (DE3) strain according to manufacturer'sinstructions (Invitrogen, Carlsbad, Calif.). Overexpression of IDH2protein was accomplished by co-transfection of expression plasmidsencoding respective IDH2 clones and pG-KJE8 expressing chaperoneproteins.

IDH1/2 Activity Assays

Enzymatic activity was assessed by following the change in NADPHabsorbance at 340 nm over time in an SFM-400 stopped-flowspectrophotometer (BioLogic, Knoxville, Tenn.) in the presence ofisocitrate and NADP+ (forward reaction), or α-KG and NADPH (reversereaction). All reactions were performed in standard enzyme reactionbuffer (150 mM NaCl, 20 mM Tris-Cl, pH 7.5, 10 mM MgCl₂ and 0.03% (w/v)bovine serum albumin) For determination of kinetic parameters,sufficient enzyme was added to give a linear reaction for 1 to 5seconds. Enzymatic binding constants were determined using curve fittingalgorithms to standard kinetic models with the Sigmaplot softwarepackage (Systat Software, San Jose, Calif.). For determination of kcat,enzyme was incubated with 5×Km of substrate and cofactor; consumption ofNADPH or NADP was determined by a change in the OD₃₄₀ over time. In bothcases an extinction coefficient of 6200 M⁻¹ cm⁻¹ was used for NADPH.

2-HG and Metabolite Analysis

Metabolites were extracted from cultured cells, primary leukemic cells,and sera using 80% aqueous methanol (−80° C.) as previously described.For cell extraction, frozen biopsies were thawed quickly at 37° C., andan aliquot of 2 million cells was spun down at 4° C. The pellet wasresuspended in −80° C. 80% methanol. For serum extraction, 1 ml of serumwas thawed quickly and mixed with 4 ml −80° C. methanol. All extractswere spun at 13000 rpm at 4° C. to remove precipitate, dried at roomtemperature, and stored at −80° C. until analysis by LC-MS. Metabolitelevels (2-HG, α-KG, succinate, fumarate, and malate) were determined byion paired reverse phase LC coupled to negative mode electrospraytriple-quadropole MS using multiple reaction monitoring, and integratedelution peaks were compared with metabolite standard curves for absolutequantification as described.

Statistical Analysis

Fisher's exact test was used to test for differences in categoricalvariables between IDH1/2 wt and IDH1/2 mutant patients. One way ANOVAfollowed by a student's t-test with correction for multiple comparisonswas used to test for differences in IDH1 activity and metaboliteconcentrations. Differences with p<0.05 were considered significant.

Results

In order to investigate the role of IDH1 R132 mutations in AML, leukemiccells obtained at initial presentation, from a series of 145 AMLpatients treated at the Princess Margaret Hospital with the aim ofidentifying mutant samples in our viable cell tissue bank weregenotyped. Heterozygous IDH1 R132 mutations were found in 11 (8%) ofthese patients (Table 25). The spectrum of IDH1 mutations observed inAML appears to differ from that seen in CNS tumors. In the CNS, themajority of mutations (80-90%) are IDH1 R132H substitutions, whereas 5,4, and 2 patients with IDH1 R132H, R132C, and R132G mutations,respectively (Table 25), were observed. In four cases, leukemic cellswere also available from samples taken at the time of relapse. The IDH1mutation was retained in 4/4 of these samples (Table 25). One of thepatients harboring an IDH1 mutation had progressed to AML from anearlier myelodysplastic syndrome (MDS). When cells from the prior MDS inthis patient were analyzed, IDH1 was found to be wild-type. Anadditional 14 patients with MDS were genotyped, and all patients werefound to be wild-type for IDH1, suggesting that IDH1 mutations are not acommon feature of this disease. In samples from a subset of IDH1 mutantpatients (n=8), reverse transcribed RNA was used for genotyping in orderto assess the relative expression of mutant and wild-type alleles.Seqenom genotyping showed balanced allele peaks for these samples,indicating that both the wild-type and mutant genes are expressed. Tenestablished AML cell lines were also genotyped (OCI/AML-1, OCI/AML-2,OCI/AML-3, OCI/AML-4, OCI/AML-5, HL-60, MV-4-11, THP-1, K562, and KG1A)and none carried an IDH1 R132 mutation. Table 25: Identification of 13AML patients bearing an IDH1 R132 or IDH2 R172 mutation*

TABLE 25 NPM1 2-HG Amino and Genotype level acid FAB FLT3 Cytogenetic at(ng/2 × 10⁶ Patient ID Mutation change subtype status profile relapsecells) IDH1 mutations 090108 G/A R132H M4 na Normal na 2090 090356 G/AR132H na na na na 1529 0034 C/T R132C M5a Normal Normal na 10285 0086C/G R132G M2 Normal Normal na 10470 0488 C/T R132C M0 Normal NormalR132C 13822 8587 G/A R132H na Normal Normal na 5742 8665 C/T R132C M1 naNormal na 7217 8741 G/A R132H M4 NPM1 Normal R132H 6419 9544 C/G R132Gna na Normal R132G 4962 0174268 G/A R132H M1 NPM1 Normal R132H 8464090148 C/T R132C M1 na 46, xx, i(7) na na (p10) [20] IDH2 mutations 9382G/A R172K M0 Normal Normal na 19247 0831 G/A R172K M1 Normal Normal na15877 NPM1 denotes nucleophosmin 1, and FLT FMS-related tyrosine kinase3. na indicates that some data was not available for some patients.

A metabolite screening assay to measure 2-HG in this set of AML sampleswas set up. Levels of 2-HG were approximately 50-fold higher in samplesharboring an IDH1 R132 mutation (Table 25, FIG. 36A, Table 26). 2-HG wasalso elevated in the sera of patients with IDH1 R132 mutant AML (FIG.36B). There was no relationship between the specific amino acidsubstitution at residue 132 of IDH1 and the level of 2-HG in this groupof patients.

TABLE 26 Metabolite concentrations in individual IDH1/2 mutant andwild-type AML cells* 2-HG α-KG Malate Fumarate Succinate IDH1/2 (ng/2 ×10⁶ (ng/2 × 10⁶ (ng/2 × 10⁶ (ng/2 × 10⁶ (ng/2 × 10⁶ Sample Genotypecells) cells) cells) cells) cells) 0034 R132C 10285 125 192 239 26510086 R132G 10470 124 258 229 3043 0488 R132C 13822 95 184 193 2671 8587R132H 5742 108 97 95 1409 8665 R132C 7217 137 118 120 1648 8741 R132H6419 87 66 61 938 9544 R132G 4962 95 76 72 1199 0174268 R132H 8464 213323 318 2287 090356 R132H 1529 138 657 366 1462 090108 R132H 2090 Na 246941 3560 090148† R132C na Na na Na Na 8741‡ R132H 2890 131 113 106 15099554‡ R132G 7448 115 208 227 2658 0174268‡ R132H 964 72 134 138 22420488‡ R132C 7511 85 289 310 3448 9382 R172K 19247 790 821 766 5481 0831R172K 15877 350 721 708 5144  157 Wild type 212 121 484 437 3057  202Wild type 121 57 161 136 1443  205 Wild type 147 39 162 153 1011  209Wild type 124 111 167 168 1610  239 Wild type 112 106 305 361 1436  277Wild type 157 61 257 257 2029  291 Wild type 113 118 124 128 1240  313Wild type 116 75 151 181 1541 090158 Wild type 411 217 658 647 3202090156 Wild type 407 500 1276 1275 6091 *IDH1/2 denotes isocitratedehydrogenase 1 and 2, 2-HG 2-hydroxy glutarate, and α-KGalpha-ketogluatarate. Metabolite measurements were not available for allpatients. †metabolic measurements were not made due to limited patientsample ‡indicates samples obtained at relapse.

Two samples harboring wild-type IDH1 also showed high levels of 2-HG(Table 25). The high 2-HG concentration prompted sequencing of the IDH2gene in these two AML samples, which established the presence of IDH2R172K mutations in both samples (Table 25).

Evaluation of the clinical characteristics of patients with or withoutIDH1/2 mutations revealed a significant correlation between IDH1/2mutations and normal karyotype (p=0.05), but no other differencesbetween these two groups (Table 27). Notably, there was no difference intreatment response for a subgroup of patients who received consistenttreatment (n=136). These findings are consistent with the initial reportidentifying IDH1 mutations in AML.

TABLE 27 Characteristics of IDH1/2 mutant and wild-type patients* IDH1/2Wild-type IDH1/2 Mutant Variable (N = 132) (N = 13) P Value Age (yr)58.8 ± 16.2 52.6 ± 7.0  0.17† Sex (% male) 53 (70/132) 62 (8/13) 0.77‡WBC at diagnosis (10⁹ cells/L) 40.7 ± 50.6 28.7 ± 34.1 0.38† Initialtreatment response 70 (85/122) 62 (8/13) 0.54‡ (% complete remission)Cytogenetic profile (% normal) 62 (72/117)  92 (11/12) 0.05‡ Additionalmutations FLT3 (%) 17 (8/47)  0 (0/8) 0.58‡ NPM1 (%) 30 (14/47)  25(2/8)  1.0‡ *For plus-minus values, the value indicates the mean, and ±indicates the standard deviation. IDH1/2 denotes isocitratedehydrogenase 1 and 2, WBC white blood cell count, FLT3 FMS-relatedtyrosine kinase 3, and NPM1 nucleophosmin 1. †P-value was calculatedusing the student's t-test. ‡P-value was calculated using Fisher's exacttest.

Panels of AML cells from wild-type and IDH1 mutant patients werecultured in vitro. There was no difference in the growth rates orviability of the IDH1 R132 mutant and wild-type cells, with both groupsshowing high variability in their ability to proliferate in culture, asis characteristic of primary AML cells (FIG. 36C). There was norelationship between 2-HG levels in the IDH1 R132 mutant cells and theirgrowth rate or viability in culture. After 14 days in culture, themutant AML cells retained their IDH1 R132 mutations (11/11), andcontinued to accumulate high levels of 2-HG (FIG. 36A), furtherconfirming that IDH1 R132 mutations lead to the production andaccumulation of 2-HG in AML cells.

To investigate the effect of IDH1/2 mutations on the concentration ofcellular metabolites proximal to the IDH reaction, α-KG, succinate,malate, and fumarate levels were measured in AML cells with IDH1/2mutations and in a set of wild-type AML cells matched for AML subtypeand cytogenetic profile. None of the metabolites were found to begreatly altered in the IDH1 mutants compared to the IDH1 wild-type cells(FIG. 27, Supplementary Table 26). The mean level of α-KG was notaltered in the IDH1/2 mutant AML cells, suggesting that the mutationdoes not decrease the concentration of this metabolite as has beenpreviously hypothesized. To confirm that the R132C mutation of IDH1, andthe R172K mutation of IDH2 confer a novel enzymatic activity thatproduces 2-HG, recombinant mutant enzymes were assayed for theNADPH-dependent reduction of α-KG. When samples were analyzed by LC-MSupon completion of the enzyme assay, 2-HG was identified as the endproduct for both the IDH1 R132C and IDH2 R172K mutant enzymes (FIG. 38).No isocitrate was detectable by LC-MS, indicating that 2-HG is the soleproduct of this reaction (FIG. 38). This observation held true even whenthe reductive reaction was performed in buffer containing NaHCO₃saturated with CO₂.

A large proportion of IDH1 mutant patients in AML have an IDH1 R132Cmutation (Table 25). In order to biochemically characterize mutant IDH1R132C, the enzymatic properties of recombinant R132C protein wereassessed in vitro. Kinetic analyses showed that the R132C substitutionseverely impairs the oxidative decarboxylation of isocitrate to α-KG,with a significant decrease in k_(cat), even though the affinity for theco-factor NADP remains essentially unchanged (Table 28). However, unlikethe R132H mutant enzyme described previously the R132C mutation leads toa dramatic loss of affinity for isocitrate (K_(M)), and a drop in netisocitrate metabolism efficiency (k_(cat)/K_(M)) of more than six ordersof magnitude (Table 28). This suggests a potential difference in thesubstrate-level regulation of enzyme activity in the context of AML.While substitution of cysteine at R132 inactivates the canonicalconversion of isocitrate to α-KG, the IDH1 R132C mutant enzyme acquiresthe ability to catalyze the reduction of α-KG to 2-HG in an NADPHdependent manner (FIG. 39). This reductive reaction of mutant IDH1 R132Cis highly efficient (k_(cat)/K_(M)) compared to the wild-type enzyme,due to the considerable increase in binding affinity of both the NADPHand α-KG substrates (K_(M)) (Table 28).

TABLE 28 Kinetic parameters of the IDH1 R132C mutant enzyme WT R132COxidative (→ NADPH) K_(M,NADP+) (μM) 49 21 K_(M,isocitrate) (μM) 57 8.7× 10⁴ K_(M,MgCl2) (μM) 29 4.5 × 10² K_(i,αKG) (μM) 6.1 × 10² 61 k_(cat)(s⁻¹) 1.3 × 10⁵ 7.1 × 10² k_(cat)/K_(M,isoc) (M⁻¹ · s⁻¹) 2.3 × 10⁹ 8.2 ×10³ Reductive (→ NADP⁺) K_(M,NADPH) (μM) n/a* 0.3 K_(M,αKG) (μM) n/a 295k_(cat) (s⁻¹) ~7 (est.) 5.5 × 10² *n/a indicates no measureable activity

We claim:
 1. A method of treating a subject having a cancercharacterized by the presence of a mutant isocitrate dehydrogenase 1enzyme (IDH1) or a mutant isocitrate dehydrogenase 2 enzyme (IDH2),wherein the mutant IDH1 or mutant IDH2 has the ability to convertalpha-ketoglutarate to 2-hydroxyglutarate (2HG), the method comprisingadministering to the subject a therapeutically effective amount of aninhibitor of said mutant IDH1 or mutant IDH2.
 2. The method of claim 1,wherein the inhibitor binds to IDH1R132X or IDH2R172X and inhibits theability to convert alpha-ketoglutarate to 2-HG.
 3. The method of claim1, wherein the cancer is characterized by an IDH1 mutation.
 4. Themethod of claim 3, wherein the IDH1 mutation is an IDH1R132X mutation.5. The method of claim 3, wherein the IDH1 mutation is selected fromR132H, R132C, R132S, R132G, R132L, and R132V.
 6. The method of claim 1,wherein the cancer is characterized by an IDH2 mutation.
 7. The methodof claim 6, wherein the IDH2 mutation is an IDH1R172X mutation.
 8. Themethod of claim 6, wherein the IDH2 mutation is selected from R172K,R172M, R172S, R172G, and R172W.
 9. The method of claim 1, wherein themutant IDH1 or mutant IDH2 is detected in a sample obtained from thesubject.
 10. The method of claim 9, wherein the sample comprises tissueor bodily fluid.
 11. The method of claim 1, wherein the mutant IDH1 ormutant IDH2 is detected by sequencing a nucleic acid from an affectedcell that encodes the relevant amino acid(s) from the mutant IDH1 ormutant IDH2.
 12. The method of claim 11, wherein the sequencing isperformed by polymerase chain reaction (PCR).
 13. The method of claim 1,wherein the inhibitor is a small molecule compound.
 14. The method ofclaim 1, wherein the cancer is selected from an astrocytic tumor, anoligodendroglial tumor, an oligoastrocytic tumor, an anaplasticastrocytoma, fibrosarcoma, paraganglioma, prostate cancer, acutelymphoblastic leukemia (ALL), and acute myelogenous leukemia (AML). 15.The method of claim 1, wherein the cancer is a glioblastoma.
 16. Themethod of claim 1, wherein the cancer is a glioma.
 17. The method ofclaim 14, wherein the cancer is AML.
 18. The method of claim 14, whereinthe ALL is B-cell ALL or T-cell ALL.